Biodefenses using triazole-containing macrolides

ABSTRACT

Use of macrolide and ketolide antibiotics for the treatment of acute exposure and diseases caused by biodefense pathogens is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national application under 37 C.F.R. §371(b)of International Application Serial No. PCT/US2009/061978 filed Oct. 24,2009, which claims priority under 35 USC §119(e) to U.S. ProvisionalApplication Ser. No. 61/108,110, filed on Oct. 24, 2008, U.S.Provisional Application Ser. No. 61/108,112, filed on Oct. 24, 2008,U.S. Provisional Application Ser. No. 61/108,134, filed on Oct. 24,2008, U.S. Provisional Application Ser. No. 61/108,137, filed on Oct.24, 2008, U.S. Provisional Application Ser. No. 61/108,168, filed onOct. 24, 2008, and U.S. Provisional Application Ser. No. 61/162,109,filed on Mar. 20, 2009, the entire disclosure of each of which areincorporated herein by reference.

TECHNICAL FIELD

The invention described herein relates to the treatment of acuteexposure and diseases caused by biodefense pathogens. In particular, theinvention described herein relates to the treatment of acute exposureand diseases caused by biodefense pathogens with macrolide and ketolideantibiotics.

BACKGROUND AND SUMMARY OF THE INVENTION

There continues to be a feared scenario of battlefield use of ordomestic terrorist attacks with aerosolized microorganisms leading tomass infections. Given the added possibility of resistance to currenttreatments through genetic engineering or natural emergence, identifyingnew effective antibiotics is critical to counter such an attack. Aneffective therapeutic agent against a spectrum of inhaled pathogens isneeded in the armamentarium of therapeutics to combat bioterror andbiowarfare. It has been surprisingly discovered that triazole-containingmacrolides and ketolides exhibit high activity on various organisms thatpose a potential biowarfare and/or bioterror threat.

In one embodiment, compounds, compositions, methods, and medicaments aredescribed herein for treating diseases arising from one or morebioterror and/or biowarfare agents. Illustrative agents include Bacillusanthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT),Burkholderia mallei (BM), and B. pseudomallei (BP). In anotherembodiment, compounds, compositions, methods, and medicaments aredescribed herein for treating diseases arising from one or morebioterror and/or biowarfare agents selected from B. anthracis, Y.pestis, F. tularensis, and B. mallei. It has been surprising discoveredherein that the triazole-containing compounds described herein arehighly active on F. tularensis. In another embodiment, the compounds,compositions, methods, and medicaments are useful as post-exposureprophylaxis agents, such as medical countermeasures, following anexposure or inhalation of a one or more bioterror and/or biowarfareagents. In another embodiment, the compounds, compositions, methods, andmedicaments are useful in treating diseases caused by an exposure orinhalation of a one or more bioterror and/or biowarfare agents,including, but not limited to, pneumonia, plague, tularemia, meliodosis,and the like.

In one illustrative embodiment, compounds of Formula (I) are describedherein

including pharmaceutically acceptable salts, hydrates, solvates, esters,and prodrugs thereof.

In one aspect, R₁₀ is hydrogen or acyl. In another aspect, X is H; and Yis OR₇; where R₇ is a monosaccharide or disaccharide, alkyl, aryl,heteroaryl, acyl, or C(O)NR₈R₉, where R₈ and R₉ are each independentlyselected from the group consisting of hydrogen, hydroxy, alkyl, aralkyl,alkylaryl, heteroalkyl, aryl, heteroaryl, alkoxy, dimethylaminoalkyl,acyl, sulfonyl, ureido, and carbamoyl; or X and Y are taken togetherwith the attached carbon to form carbonyl.

In another aspect, V is C(O), C(═NR₁₁), CH(NR₁₂, R₁₃), or N(R₁₄)CH₂,where N(R₁₄) is attached to the C-10 carbon of the compounds of Formulae1 and 2; wherein R₁₁ is hydroxy or alkoxy, R₁₂ and R₁₃ are eachindependently selected from the group consisting of hydrogen, hydroxy,akyl, aralkyl, alkylaryl, alkoxy, heteroalkyl, aryl, heteroaryl,dimethylaminoalkyl, acyl, sulfonyl, ureido, and carbamoyl; R₁₄ ishydrogen, hydroxy, alkyl, aralkyl, alkylaryl, alkoxy, heteroalkyl, aryl,heteroaryl, dimethylaminoalkyl, acyl, sulfonyl, ureido, or carbamoyl.

In another aspect, W is H, F, Cl, Br, I, or OH.

In another aspect, A is CH₂, C(O), C(O)O, C(O)NH, S(O)₂, S(O)₂NH,C(O)NHS(O)₂. In another aspect, B is (CH₂). where n is an integerranging from 0-10, or B is an unsaturated carbon chain of 2-10 carbons.In another aspect, C is hydrogen, hydroxy, alkyl, aralkyl, alkylaryl,alkoxy, heteroalkyl, aryl, heteroaryl, aminoaryl, alkylaminoaryl, acyl,acyloxy, sulfonyl, ureido, or carbamoyl.

In another embodiment, compositions including a therapeuticallyeffective amount of one or more compounds of formula (I), or the varioussubgenera thereof are described herein. The pharmaceutical compositionsmay include additional pharmaceutically acceptable carriers, diluents,and/or excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Preliminary in vivo studies in animal models of disease alsodemonstrate protection against these pathogens.

FIG. 2. CEM-101 Minimum Inhibitory Concentration Distributions are shownin bar graph form

FIG. 3. Comparative susceptibilities of S. aureus ATCC 25923 and L.monocytogenes EGD to CEM-101, TEL, AZI, and CLR, based on MICdeterminations in pH-adjusted broth.

FIG. 4. Short-term time-kill effect of CEM-101 and AZI on S. aureus(ATCC 25923) in broth (left panels; pH 7.4) or after phagocytosis byTHP-1 macrophages (right panels). Both drugs were used at anextracellular concentration of either 0.7 (top panels) or 4 (bottompanels) mg/liter. MICs of CEM-101 and AZI were 0.06 and 0.5 mg/liter,respectively. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 5. Concentration-effect relationships for CEM-101, TEL, CLR, andAZI toward S. aureus (ATCC 25923) in broth (left panels) and afterphagocytosis by THP-1 macrophages (right panels). The ordinate shows thechange in CFU (Δ log CFU) per ml (broth) or per mg of cell protein(THP-1 macrophages) at 24 h compared to the initial inoculum. Theabscissa shows the concentrations of the antibiotics as follows: (i) toppanels, weight concentrations (in mg/liter) in broth (left) or in theculture medium (right) and (ii) bottom panels, multiples of the MIC asdetermined in broth at pH 7.4. All values are means±standard deviations(SD) of three independent experiments (when not visible, SD bars aresmaller than the symbols). Statistical analysis based on global analysisof curve-fitting parameters (one-way analysis of variance); the onlysignificant difference is between CEM-101 and AZI in broth (P=0.04).Numerical values of the pertinent pharmacological descriptors andstatistical analysis of their differences are shown in Table 1.

FIG. 6. Concentration-effect relationships for CEM-101 and AZI towardintraphagocytic L. monocytogenes (strain EGD, left panels) and L.pneumophila (strain ATCC 33153, right panels). The ordinate shows thechange in CFU (Δ log CFU) per mg of cell protein at 24 h (L.monocytogenes) or 48 h (L. pneumophila) compared to the initialpostphagocytosis inoculum. The abscissa shows the concentrations of theantibiotics as follows: (i) top panels, weight concentrations (inmg/liter); (ii) bottom panels, multiples of the MIC as determined inbroth at pH 7.4. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 7. Accumulation of CEM-101 versus comparators in THP-1 cells at 37°C. (all drugs at an extracellular concentration of 10 mg/liter). (A)Kinetics of accumulation (AZI); Cc, intracellular concentration; Ce,extracellular concentration); (B) influence of the pH of the culturemedium on the accumulation (30 min) of CEM-101 (solid symbols and solidline) and AZI (open symbols and dotted line); (C) influence of monensin(50 μM; 2-h incubation), verapamil (150 μM; 24-h incubation), orgemfibrozil (250 μM; 24-h incubation) on the cellular accumulation ofAZI and CEM-101. All values are means±standard deviations (SD) of threeindependent determinations (when not visible, SD bars are smaller thanthe symbols).

FIG. 8. Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages. Bars represent the MICs (in mg/L) or theextracellular static dose.

FIG. 9. Intracellular Activity of CEM-101 compared to AZI, CLR, and TEL,expressed as a dose response curve of Δ log CFU from time 0 to 24 hoursversus log dose.

DETAILED DESCRIPTION

In one embodiment, compounds are described herein that are activeintracellularly. It has also been discovered herein that theintracellular accumulation and intracellular activity oftriazole-containing macrolides was not affected by Pgp or MultidrugResistant Protein (MRP) inhibitors. Accordingly, it is believed that thecompounds described herein are not substrates or are poor substrates ofP-glycoprotein (plasma or permeability gycoprotein, Pgp). It isappreciated that Pgp is an efflux mechanism that may lead to resistanceby some organisms against certain antibiotics, such as has been reportedfor AZI and ERY in macrophages in which both antibiotics are substratesof the P-glycoprotein. Accordingly, it has been surprisingly found thatthe compounds described herein accumulate intracellulary. In addition tothe intracellular accumulation, it has been surprisingly discovered thatthe triazole-containing macrolide and ketolide compounds describedherein have high intracellular activity. It has also been surprisingfound herein that the compounds described herein have lower proteinbinding than is typical for macrolides at lower pH, such as the pH foundin bacterial infections, including but not limited to abscesses. It isappreciated that the lack of intracellular activity typically observedwith anti-bacterial agents, including other macrolides and ketolides,may be due to high protein binding, and/or to the relatively lower pH ofthe intracellular compartments, such as is present in abscesses.

However, even when not removed by active efflux, the concentration ofother anti-bacterial agents, including other macrolides and ketolides,in macrophages may not be efficacious in treating disease because of thelow pH of the lysozomal compartment. For example, the acidic environmentprevailing in the phagolysosomes (where one or more of B. anthracis, Y.pestis, F. tularensis, and/or B. mallei may sojourn during itsintracellular stage) may impair the activity of antibiotics, such as theAZI, CLR and TEL. It has been unexpectedly found that the compoundsdescribed herein retain their anti-bacterial activity at low pH. It isappreciated that the intracellular activity of the compounds describedherein may be an important determinant for fast and complete eradicationand, probably also, for prevention of resistance in the target organism.

Lack of effective antimicrobial therapy results in intracellularsurvival of bacteria, which remains a major cause of bacterialspreading, life-threatening therapeutic failures, and establishment ofchronic, relapsing infections. These situations are observed during thecourse of infections caused by many biodefense organisms, including B.anthracis, Y. pestis, F. tularensis, and B. mallei.

While it has been reported that intracellular accumulation of anantibiotic is indicative of efficient activity against bacteria,pharmacodynamic evaluation of a large series of commonly usedantibiotics has revealed that other parameters such as intracellularbioavailability and modulation of activity in the infected compartmentare also important. The observations described herein confirm and extendprevious observations made with macrolides in this context due to thesurprising differential behavior exhibited by the triazole-containingmacrolides described herein, compared to known macrolide and ketolides ,such as TEL, AZI, and CLR.

It is surprisingly found that triazole-containing macrolides accumulateto a considerably larger extent than the comparators, including AZI, andconsistently expresses greater potency (decreased values of E₅₀ andC_(s)) while showing similar maximal efficacy (E_(max)) to comparators.Without being bound by theory, it is believed that this indicates thatthe improvements resulting from the structural modifications introducedin CEM-101 relate to modulation of pharmacokinetic properties andintrinsic activity (including its reduced susceptibility tophysico-chemical conditions prevailing in the infected compartment)rather than to a change in its mode of action. Thus, triazole-containingmacrolides exhibit the essentially bacteriostatic character ofmacrolides, but express it better in the intracellular milieu and atconsiderably lower extracellular concentrations than the comparators.

Without being bound by theory, it is believed that the cellularaccumulation of triazole-containing macrolides, such as CEM-101, resultsfrom the general mechanism of proton trapping of weak organic basesenvisaged for all macrolides as accumulation is almost completelysuppressed, in parallel with AZI, by exposure to acid pH or to theproton ionophore monensin. Based on the general model ofdiffusion/segregation of weak bases in acidic membrane-boundcompartments, accumulation is determined by the number of ionizablegroups and the ratios between the membrane permeability coefficients ofthe unionized and ionized forms of the drug. While CEM-101 has twoionizable functions, the pKa of the aminophenyltriazole is calculated tobe less than 4, suggesting that the molecule is largely monocationic(similar to CLR and TEL) at neutral and even at lysosomal pH (˜5). Incontrast, AZI has two ionizable functions with pK_(a)s>6 and istherefore dicationic intracellularly. CEM-101, however, possesses afluoro substituent in position 2, which should make it more lipophilicthan CLR or TEL. Without being bound by theory, it is believed that theratio of the permeability constants of the unionized and ionized formsof CEM-101 in comparison with LR or TEL may be as important as thenumber of ionizable functions to determine the level of cellularaccumulation of weak organic bases. Without being bound by theory, it isbelieved that the greater cellular accumulation of CEM-101 may bepartially due to its lack of susceptibility to Pgp-mediated efflux(which is expressed by THP-1 macrophages under our culture conditions)in contrast to azithromycin and other macrolide or ketolide antibiotics.

It has been observed that many known macrolides have a large volume ofdistribution, which it is believed is related to their ability toaccumulate inside eukaryotic cells by diffusion/segregation in acidiccompartments, namely lysosomes and related vacuoles. As a consequence,known macrolides had been considered candidates for the treatment ofinfections localized in these compartments. Thus, it might be assumedthat macrolides are suitable for treating infections caused by typicalintracellular pathogens such as B. anthracis, Y. pestis, F. tularensis,and B. mallei. However, direct quantitative comparisons betweenintracellular and extracellular activities using facultativeintracellular pathogens, such as S. aureus or L. monocytogenes, suggestthat known macrolides express only a minimal fraction of theirantibacterial potential intracellularly, especially considering theirgreat intracellular accumulation. This minimized antibacterial potentialagainst organisms replicating in phagolysosomes and related vacuoles isbelieved to be related to acidic pH which is known to reduce theactivity of known macrolides. Another factor is that some organisms,such as B. anthracis, Y. pestis, F. tularensis, and B. mallei, mayactually replicate in other subcellular compartments. In addition,certain macrolides, such as AZI, are subject to active efflux frommacrophages, which further contributes to suboptimal intracellularactivity.

In contrast, the cellular accumulation and intracellular activity of thetriazole-containing compounds described herein, using models that havebeen developed for the study of the intracellular pharmacodynamics ofantibiotics, is substantially improved over known macrolides, includingketolides. Thus, the compounds described herein maintain the maximalefficacy of their MICs, and show greater potency against intracellularforms of for example, B. anthracis, Y. pestis, F. tularensis, and B.mallei compared to TEL, AZI, and CLR. Without being bound by theory, itis believed that this improved intracellular potency of thetriazole-containing compounds described herein results from thecombination of the higher intrinsic activity against B. anthracis, Y.pestis, F. tularensis, and B. mallei coupled with the retained activityat low pH, and the ability to distribute to a wide variety ofintracellular compartments.

In another embodiment, the triazole-containing macrolide and ketolidecompounds have intracellular activity, such as intracellular activityagainst B. anthracis, Y. pestis, F. tularensis, and B. mallei. Survivalof these organism within eukaryotic cells is critical for thepersistence of infection. It is appreciated that routine susceptibilitytesting are usually determined against extracellular bacteria only, andtherefore may be misleading in their prediction of efficacy againstintracellular organisms.

In another embodiment, the compounds, methods, and medicaments describedherein include a therapeutically effective amount of one or morecompounds described herein, wherein the therapeutically effective amountis an amount effective to exhibit intracellular antibacterial activity.

In another embodiment, compounds are described herein that arebactericidal. In another embodiment, the compounds, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidalactivity, including in vivo bactericidal activity. It has been reportedthat macrolides are generally bacteriostatic. Bacteriostatic compoundsdo not kill the bacteria, but instead for example inhibit growth andreproduction of bacteria without killing them; killing is accomplishedby bactericidal agents. It is understood that bacteriostatic agents mustwork with the immune system to remove the microorganisms from the body.Bacteriostatic antibiotics may limit the growth of bacteria via a numberof mechanisms, such as by interfering with bacterial protein production,DNA replication, or other aspects of bacterial cellular metabolism. Incontrast, bactericidal antibiotics kill bacteria; bacteriostaticantibiotics only slow their growth or reproduction. Several bactericidalmechanism have been reported, including disrupting cell wall precursorleading to lysis, binding irreversibly to 30s ribosomal subunit,reducing translation fidelity leading to inaccurate protein synthesis,and inhibit protein synthesis due to premature separation of the complexbetween mRNA and ribosomal proteins. The final result is bacterial celldeath.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidal activityagainst one or more of B. anthracis, Y. pestis, F. tularensis, and B.mallei. Without being bound by theory, it is believed herein thattreating such diseases using bacteriostatic agents may be unsuccessfulin two respects. First, simply stopping the progression of the diseasewith a bacteriostatic agent may be insufficient because the immunesystem may not intervene to assist in curing the disease at a necessarylevel. For example, some bacterial organisms are not killed by theimmune system because they reside in intracellular compartments. Thus,once the treatment course has ended, rapid recurrence of disease mayresult. Second, because some portion of the bacterial population willlikely be eliminated, the remaining population may be selected forresistance development. It is believed herein that an intracellularlyactive agent, and/or an intracellularly active and bactericidal agent,will be efficacious in treating such diseases. In one illustrativeembodiment, compounds described herein that achieve an intracellularconcentration of 20× the MIC of the targeted bacteria. It has beenreported that most, if not all, macrolide antibiotics, thoughbactericidal in vitro, are only bacteriostatic in vivo. For example, asdescribed herein, when the time between the last dose of compound wasextended, the bioload reduction levels remained the same for thetriazole-containing compounds described herein, indicating abactericidal response. In contrast, the TEL and CLR dose groupsdemonstrated bioload increases when the time interval was extended.Thus, those latter two macrolide/ketolide agents demonstrated a moreclassical bacteriostatic response.

CEM-101 was also found to have potent in vitro activity against B.anthracis which compares favorably with that of antibiotics currentlyapproved (ciprofloxacin, MIC₉₀ 0.031 ug/ml) or proposed (cethromycin,MIC₉₀ 0.063 ug/ml) for post-exposure anthrax indications.

As shown in FIG. 1, dose-dependent activity (percent survival) againstaerosolized B anthracis is observed over 14 days of oral dosing. Ofparticular significance is the fact that the protective doses shown here(e.g. 2.5-20.00 mg/kg) also provide high serum and tissue levels ofCEM-101 in rodent models, levels which are achievable with safe dosingregimens in human trials. Based on accrued GLP toxicology data, itshould be possible to administer CEM-101 for longer intervals (e.g.30-60 days) as recommended for management of post exposure inhalationalanthrax. (Drusano, et al., Antimicrobial Agents and Chemotheraspy,November, 2008, p3973-3979, Vol 52, No 11.) These prolonged prophylacticregimens are required to eradicate vegetative cells followinggermination of dormant spores, known to exist for a variable andpotentially extended duration in pulmonary tissues of exposed subjectsprior to development of clinical symptoms (Inglesby, et al., JAMA 2002;287(17) 2236-2252). Moreover, given the uncertainties about duration ofspore latency after exposure or even after discontinuation of a 30-60day prophylactic regimen, acute therapy with an oral formulation may berequired in selected symptomatic subjects in this biodefense scenario.

In cellular models, CEM-101 is significantly more active than otherantibacterial agents against organisms located intracellularly. It isactive against resistant bacteria, including multi-drug resistantbacteria. Evidence of bacterial resistance to CEM-101 in vitro isobserved. Without being bound by theory, it is believed that thoseexamples that become resistant are likely not to have a survivaladvantage as they would have multiple mutations that decrease viabilityand virulence

The capacity of CEM-101 to accumulate in tissues and to achieve highintracellular concentrations, with antimicrobial potency, is apharmacologic characteristic, because intracellular parasitism underliesthe pathophysiology of disease due to the biothreat agents of concernhere. FIG. 6 shows that macrophage uptake and intracellular killing ofL.egionella pneumophila (lysosomal compartment) and Listeriamonocytogenes (cytoplasm) by CEM-101 is even more active than by othermacrolide agents tested (Lemaire, et al., Antimicrob. Agents. Chemother.53: 3734-3743, 2009). Intracellular concentrations that are 20-200 foldhigher than achieved in plasma is facilitated by rapid uptake into thecell and evasion of the efflux pump P-glycoprotein, thus allowingeffective eradication of replicating intracellular pathogens. CEM-101,unlike azithromycin and cethromycin, is not a substrate forP-glycoprotein.

Several in vivo protocols wherein CEM-101 was repeatedly dosed intoxicology studies in rodents and non human primates have demonstratedtissue levels of CEM-101˜17-100× higher than peak plasma levels. CEM-101accumulated in tissues and concentrations were highest in liver, spleen,lung, and salivary gland. This relationship was confirmed in rodent ADMEstudies using radiolabeled CEM-101. When administered orally at 100mg/kg, lung tissue to plasma radioactivity ratios of ˜13:1 were observedin male and female animals. After IV dosing at 20 mg/kg, the data wasmore variable and lung/plasma ratios of 17.6 for males and 6.2 forfemales were observed. Cmax and AUC ranged from 0.022 μg/mL and 0.04μg·h/mL to 1.96 μg/mL and 28.60 μg·h/mL across the dose range. The meanCEM-101 t_(max) increased from 1.5 to 6.0 hours and the mean terminalhalf-life increased from 2.2 to 7.9 hours over the 50 to 1600 mg doserange.

In another illustrative embodiment, compounds of Formula (I) aredescribed herein where X and Y are taken together with the attachedcarbon to form a C(O) group. In another embodiment, X is H, Y is OR⁷,where R⁷ is a monosaccharide radical, such as cladinosyl. In anotherembodiment, compounds of Formula (I) are described herein where W isfluoro. In another embodiment, compounds of Formula (I) are describedherein where A and B are taken together to form an alkylene group,including but not limited to propylene, butylene, and pentylene. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form butylene. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene. In another embodiment, compounds of Formula(I) are described herein where A and B are taken together to formbutylenes and C is 2-pyridinyl or aminophenyl, such as 3-aminophenyl. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form propylenes, butylenes, or pentylenes;and C is aminophenyl, such as 3-aminophenyl. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene and C is 3-pyridinyl or benzotriazole. Inanother embodiment, compounds of Formula (I) are described herein whereC is an optionally substituted aryl or heteroaryl group. In anotherembodiment, compounds of Formula (I) are described herein where V is acarbonyl group. In another embodiment, compounds of Formula (I) aredescribed herein where R¹⁰ is hydrogen. In another embodiment, X is H, Yis OR⁷, where R⁷ is a monosaccharide radical, such as cladinosyl, and Cis 3-pyridinyl or benzotriazolyl.

In another embodiment, C is optionally substituted phenyl, such asphenyl, halophenyl, haloalkylphenyl, aminophenyl, and the like,optionally substituted pyridinyl, such as 2-pyridinyl and 3-pyridinyl,optionally substituted benzotriazole, and the like.

In another embodiment, A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group.

In another embodiment, compounds described in any of the precedingembodiments wherein V is C(O) are described. In another embodiment,compounds described in any of the preceding embodiments wherein W is Hor F are described. In another embodiment, compounds described in any ofthe preceding embodiments wherein A is CH₂, B is (CH₂)_(n), and n is aninteger from 2-4 are described. In another embodiment, compoundsdescribed in any of the preceding embodiments wherein C is aryl orheteroaryl are described. In another embodiment, compounds described inany of the preceding embodiments wherein C is 3-aminophenyl or3-pyridinyl are described. In another embodiment, compounds described inany of the preceding embodiments wherein R₁₀ is hydrogen. In anotherembodiment, compounds described in any of the preceding embodimentswherein A and B are taken together to form butylene or pentylene, and Xand Y are taken together with the attached carbon to form a C(O) group.In another embodiment, compounds described in any of the precedingembodiments wherein A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group, and W is F.

In another embodiment, an antibacterial composition is described herein,wherein the composition includes an effective amount of one or morecompounds described herein, and a pharmaceutically acceptable carrier,excipient, or diluent therefor, or a combination thereof.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. Illustratively,compositions may include one or more carriers, diluents, and/orexcipients. The compounds described herein may be formulated in atherapeutically effective amount in conventional dosage forms for themethods described herein, including one or more carriers, diluents,and/or excipients therefor. Such formulation compositions may beadministered by a wide variety of conventional routes for the methodsdescribed herein in a wide variety of dosage formats, utilizingart-recognized products. See generally, Remington's PharmaceuticalSciences, (16th ed. 1980). It is to be understood that the compositionsdescribed herein may be prepared from isolated compounds describedherein or from salts, solutions, hydrates, solvates, and other forms ofthe compounds described herein. It is also to be understood that thecompositions may be prepared from various amorphous, non-amorphous,partially crystalline, crystalline, and/or other morphological forms ofthe compounds described herein.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known in the medical arts.

In one embodiment, the compounds described herein are administered to ahuman orally at a dose of about 1 to about 10 mg/kg, about 2 to about 8mg/kg, or about 4 to about 6 mg/kg of patient body weight. In anotherembodiment, the daily adult human dose is about 100 to about 1,000 mg,which may be administered qd, bid, tid, and the like. In anotherembodiment, the daily adult human dose is about 400 to about 600 mg,which may be administered qd, bid, tid, and the like. Such doses may beadministered, once, twice, or thrice per day. Illustrative oral unitdosages are 50, 100, 200, and 400 mg (single or divided). Without beingbound by theory, it is believed that such illustrative dosages aresufficient to achieve plasma levels of about 1 μg/mL, which may besufficient to observe bactericidal activity of the compounds describedherein, such as for one or more of B. anthracis, Y. pestis, F.tularensis, and B. mallei. It is appreciated that as described herein,the compounds described herein, including CEM-101, reach highconcentration in tissues, such as lung tissues. Without being bound bytheory, it is believed herein that the compounds described herein,including CEM-101, may achieve tissue levels that are at least about10-times the MIC for strains, including macrolide-resistant strains,such as but not limited to B. anthracis, Y. pestis, F. tularensis, andB. mallei, including resistant strains thereof.

The compounds described herein may be prepared as described herein, oraccording to US Patent Application Publication No. 2006/0100164 and inPCT International Publication No. WO 2009/055557, the disclosures ofwhich are incorporated herein by reference in their entirety.

Briefly, the synthesis of triazole containing ketolides begins with theknown two step preparation of the 12-acyl-imidazole intermediate 4(Scheme I) from clarithromycin (2). Intermediate 4 is converted into the11,12-cyclic carbamates 5a-c by the reaction with the corresponding 3-,4- or 5-carbon linked amino alcohols. Treatment of 5a-c with tosylchloride provides tosylates 6a-c. Displacement of the tosyl group withNaN₃ gives the corresponding azido compounds 7a-c. Cleavage of thecladinose sugar of 7a-c to 8a-8c is accomplished by treatment with HClin MeOH. Swern oxidation of the 3-hydroxy group of 8a-c gives thecorresponding protected ketolides 9a-c which are subsequentlydeprotected with methanol to afford the required azido ketolides 10a-c,respectively. These azido compounds were reacted withterminally-substituted alkynes in the presence of copper iodide intoluene at 60° C. to regio-selectively afford the corresponding4-substituted-[1,2,3]-triazoles 11a-18a, 11b-18b, and 11c-18c.

The azide of intermediates 10a-c is converted to the4-substituted-[1,2,3]-triazoles via a cycloaddition reaction withsubstituted acetylenes. Triazole rings may be formed via a Huisgen 1+3cycloaddition reaction between an azide and an alkyne resulting in amixture of 1,4- and 1,5-regioisomers as depicted in Route A of SchemeII. Alternatively. the procedure of Rostovtsev et al.⁸ may be followedusing the addition of a CuI catalyst to the reaction to selectively orexclusively produce the 1,4-regioisomer as depicted in Route B of SchemeII.

The triazole ring side chain is also incorporated into theclarithromycin ring system. In one embodiment, a butyl alkyl side chainis chosen. It is appreciated that many butyl side chain analogs in theketolide series have improved antibacterial activity based on in vitroMIC results. Intermediate 7b is directly converted into the4-substituted-[1,2,3]-triazole via copper catalyzed cyclization withterminally substituted acetlyenes, as shown in Scheme III. The acetateprotecting groups of 19a-e are removed with LiOH in methanol to affordthe corresponding 4-substituted-[1,2,3]-triazoles 20a-e.

Substitution of the 2- position hydrogen with a fluorine is accomplishedby electrophilic fluorination of 9b (Scheme IV) using Selectfluor®. Theazido group of intermediate 22 is converted to a series of4-substituted-[1,2,3]-triazoles 23a-b via the standard conditions.

In another embodiment, the following compounds are described:

Minimum inhibitory concentration (μg/mL)^(a) S. aureus S. pneumoniae H.influenzae 29213 96:11480 49619 163 Ery-R 303 Ery-R 49247 Entry R nEry-S Ery-R (MLSb) Ery-S (MefA) (ermB) Ery-S TEL ≦0.125  ≦0.125 ≦0.125 ≦0.125  ≦0.125    4 AZI ≦0.125 >64 ≦0.125 >64 >64    2 11a 11b 11c

3 4 5    1 ≦0.125 ≦0.125     1     0.25  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125 >64     2     0.25 >64    8   16 12a 12b 12c

3 4 5    0.25 ≦0.125 ≦0.125     0.5  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125     8     8     1   64    8   16 13a 13b 13c

3 4 5    1    0.25    0.5     2     0.25     1 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125     0.5   16     8     2 >64    8   64 14a 14b 14c

3 4 5    2 ≦0.125 ≦0.125     2  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125    0.5  ≦0.125  ≦0.125 >64  ≦0.125     0.25 >64    4   64 15a 15b 15c

3 4 5    2 ≦0.125 ≦0.125     2     4     0.25 ≦0.125 ≦0.125 ≦0.125     1    2     0.25 >64   64    4 >64   64   16 16a 16b 16c

3 4 5    0.5 ≦0.125 ≦0.125 nt  ≦0.125  ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125  ≦0.125  ≦0.125 >64  ≦0.125     0.25   16    2    8 17a 17b 17c

3 4 5    1 ≦0.125    0.25     1  ≦0.125     0.5 ≦0.125 ≦0.12 ≦0.125 ≦0.125  ≦0.12  ≦0.125 >64     1     2 >64   16   32 18a 18b 18c

3 4 5    1    1 ≦0.125     2     2  ≦0.125 ≦0.125 ≦0.125 ≦0.125     0.5    4  ≦0.125 >64    64    64 >64   32    8 ^(a)National Committee forClinical Laboratory Standards. Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically, 6^(th) ed.;Approved standard: NCCLS Document M7-A6, 2003.

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae H. influenzae 25923 49619 163 Ery-R 303 Ery-R49247 Entry R Ery-S RN220 Ery-S (MefA) (ermB) Ery-S TEL ≦0.25 2 ≦0.125≦0.125 ≦0.125 4 20a

0.25 8 ≦0.0625 0.125 2 NT 20b

0.25 8 ≦0.0625 ≦0.06 1 NT 20c

1 8 ≦0.0625 0.5 2 NT 20d

1 8 ≦0.0625 0.5 2 NT 20e

≦0.25 58 ≦0.0625 0.5 2 NT

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae H. influenzae 29213 96:11480 49619 163 Ery-R 303Ery-R 49247 Entry R Ery-S Ery R (MLSb) Ery-S (MefA) (ermB) Ery-S TEL≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 4 AZI ND ≦0.125 >64 ≦0.125 >64 >6423a

≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2 23b (CEM-101)

≦0.06 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2

In each of the foregoing embodiments, the primary screening panelconsisted of relevant Staph. aureus, S. pyogenes, S. pneumoniae(including strains resistant to azithromycin and telithromycin). MICsagainst all pathogens were determined using broth microdilution methodas per NCCLS guidelines. Compounds described herein, such as CEM-101were found to be highly potent having MICs against S. pneumoniae (3773)of ≦0.125 μg/mL and S. pyogenes (1850) of 0.5 μg/mL, compared to 1 and 8μg/mL, respectively for Telithromycin. CEM-103 (20c), an analogue ofCEM-101 that contains the 3-O-cladinose was found to be less active.Non-heteroaromatic substituted triazole containing ketolides were lessactive.

The ketolides were tested against erythromycin-sensitive (Ery-S) anderythromycin-resistant (Ery-R) strains of S. aureus (29213 (Ery-S) and96:11480 (Ery-R)), S. pneumoniae (49619 (Ery-S) and 163 and 303 (Ery-R))and H. influenzae (49247 (Ery-S)) (Tables 1-3). The broth micro-dilutionmethod was used to determine the Minimum Inhibitory Concentrations(MICs) against all pathogens as per the Clinical and LaboratoryStandards Institute (CLSI).

The chain length of the alkyl side chain had a affected activity (Table1). For example, the 3-carbon linked phenyl substituted triazole 11a wasless active against Ery-S and Ery-R S. aureus and was inactive againstEry-R S. pneumoniae 303 (ermB) a the tested concentrations, whereas thecorresponding 4- and the 5-carbon linked phenyl substituted triazoles11b and 11c were more active against these organisms. A similar trendwas observed for the 2-pyridyl substituted triazoles 14a-c, the3-amino-phenyl substituted triazoles 16a-c, and the 2,5-dichlorophenoxysubstituted triazoles 17a-c.

The 4-carbon linked 2-pyridyl substituted triazole 14b and the3-amino-phenyl substituted triazole 16b possessed the highest potencyagainst S. pneumoniae 303, both having MIC values (≦0.125 μg/mL)comparable to telithromycin. The ketolide containing the 4-carbon linked3-pyridyl substituted triazole 15b was less active against this strain(MIC of 64 μg/mL). Within this series antibacterial activity wasimproved by extending the carbon linker to 5 atoms, for example the MICagainst S. pneumoniae 303 for compound 15c improved from 64 to 4 μg/mL.A similar effect was also observed for the benzo-triazole containingketolide 18c against S. aureus but 18c was still inactive against S.pneumoniae 303. It is appreciated that a balance between the length ofthe linker and nature of the aromatic substitution of the triazole mayaffect the overall acitivity against macrolide resistant S. pneumoniaand S. aureus.

A correlation between linker length and activity was also observed forH. influenzae (49247) where the most potent ketolide series had thesubstituted triazole linked through either a 4-carbon (11b-14b, 16b,17b) or a 5-carbon (15c, 18c) chain. Interestingly, the most potentaromatic series against H. influenzae was the 3-amino-phenyl with a 3-,4- or 5-carbon linker (16a, 16b, 16c) having MICs of 16, 2, and 8 μg/mL,respectively,

The macrolides containing a cladinose at the 3 position were all highlyactive against Ery-S S. pneumoniae (49619) (Table 2). However, theseanalogs were less potent than telithromycin against Ery-R strains. TheMICs were significantly higher for the cladinose containing analogs witheither 2-pyridyl, 2-aminophenyl or 2,6-dichlorophenyl triazolesubstituents than for the corresponding ketolides (20a, 20c, and 20dversus 14b, 16b, and 17b). Conversely, antibacterial activity wasre-established for ketolide analogs 15b (3- pyridyl) and 18b(benzo-triazole) by replacing the keto with the cladinose group inanalogs 20b (3- pyridyl) and 20e (benzo-triazole). The MICs improvedfrom 64 μg/mL for 15b and 18b to 1 and 2 μg/mL for 20b and 20e,respectively. A similar activity trend was also observed for Ery-R S.pneumoniae 163 (MefA).

COMPOUND EXAMPLES

A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-(3-dimethylamine-4-deoxy-6-O-acetyl-glu-copyranosyl)-2-fluoro-3-oxo-erythronolideA, 11,12-carbamate (15 mg, 0.019 mmol), 6-Ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform: methanol plus1% ammonium hydroxide) to give 14 mg of the desired compound. MS:C₄₄H₆₆FN₇O₁₂ calculated M⁺=903.5, Found: M+H⁺=904.5.

11-N-4-(3-aminophenyl)-[1,2,3]triazol-1-yl]-butyl}-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate (CEM-101). A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (17 mg, 0.023 mmol), 3-Ethynyl-phenylamine (5.4 mg,0.046 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heated to70° C. After 16 h, the mixture was concentrated and directly subjectedto silica gel chromatography (9:1, chloroform:methanol plus 1% ammoniumhydroxide) to give 17 mg of the desired compound, MS C₄₃H₆₅FN₆O₁₀calculated M⁺=844.47, Found: M+H⁺=845.5

11-N-{4-[4-(6-Amino-pyridin-2-yl)-[1,2,3]triazol-1-yl]-butyl}-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (15 mg, 0.02 mmol), 6-ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 14 mg of the desired compound OP1357. MS:C₄₂H₆₄FN₇O₁₀ calculated M⁺=845.5, Found: M+H⁺=846.5.

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-O-dasosaminyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-O-desosaminyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.0039 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₀ calculated M⁺=866.5, Found: M+H⁺867.5

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-mycaminosyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-mycaminosyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.004 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₁ calculated M⁺=882.5, Found: M+H⁺=883.5.

METHOD EXAMPLES Example

Pivotal Efficacy of CEM-101 against a Lethal Inhalational B. anthracisChallenge in Cynomolgus Macaques evaluating the efficacy of CEM-101against a lethal aerosol challenge. Forty-two (21 male, 21 female) naïveCynomolgus macaques approximately 2.5-5.0 kg, ˜2-5 years of age,(available from Covance) are tested (40 placed on study and 2 extras).Monkeys are tested and verified negative for tuberculosis and alsoprescreened within 30 days prior to receipt to confirm that they areseronegative for Simian Immunodeficiency Virus (SW), SimianT-Lymphotrophic Virus-1 (STLV-1), and Cercopithecine herpesvirus 1(Herpes B virus) and negative for Simian Retrovirus (SRV1 & SRV2) byPCR. See Table 7.

TABLE 7 Proposed Study Design Time to Treatment Group Monkeys/Antibiotic Dose Dosing 1st Dose Duration ID Group or Vehicle (mg/kg)Regimen (hrs PC) (days) 1 10 CEM-101  5 QD 24 14 2 10 CEM-101 10 QD 2414 3 10 CEM-101 15 QD 24 14 4 10 Sterile 2 mL/kg QD 24 14 Water forInjection

Animals are weighed on study days −7 and 0. Monkeys that die or areeuthanized on study are weighed prior to necropsy. Additional weightsmay be taken if an animal appears to be losing weight during the courseof disease progression. Clinical observations of all monkeys performedtwice daily during the pre-challenge period. Blood samples are takenfrom a femoral artery or vein, saphenous vein, or other appropriate veinon the days specified in Table 8. Blood samples collected on the day ofchallenge are collected prior to challenge. Body temperatures aremonitored via an implantable programmable temperature transponder chip(such as the IPTT-300 BMDS, Seaford, Del.) twice a day starting atimplantation (˜day −7) through day 28.

TABLE 8 Blood Draw Schedule Bac- Coagula- teremia CBC/ ClinicalAntibiotic tion Time Point (Culture) CRP Chem. Level Det. Assay Day −7 XX X X Day 0 X X X (prior to challenge) 24 hours PC X X X X (Prior totreatment) 28 hours PC X (~4 hours PT) 48 hours PC X X X X X 72 hours PCX X X X 96 hours PC X X X X Day 7 X X X X X (~4 hours PT) Day 8 X Day 14X X X X X (~4 hours PT) Day 15 X Day 21 X X X X Day 28 X X X X Day 30 XDay 32 X Terminal X CRP PT = post-treatment, PC = post-challenge

Monkeys are transported into the BL (biohazard laboratory level 3)—˜5-10days prior to challenge to allow time for acclimation. Monkeys withineach challenge day group are randomized for challenge order prior tochallenge. On day 0, monkeys are anesthetized with Telazol (1-6 mg/kg,IM) and placed into a plethysmography chamber and a Class III cabinetsystem for the targeted challenge agent aerosolized by a Collisonnebulizer and delivered via a head-only inhalation exposure chamber.

Aerosol concentrations of challenge material are quantified bydetermination of colony forming units (cfu). Effluent streams arecollected directly from an animal exposure port by an in-line all-glassimpinger. Serial dilutions of impinger samples are plated and counted.

Following challenge, monkeys are observed twice daily for a minimum of28 days under BL-3 conditions for survival and clinical signs ofillness. Surviving animals may be removed from the BL-3 after day 28subsequent to demonstrating three consecutive negative blood cultures.

Blood samples from time points indicated in Table 8 are cultured todetermine the presence or absence of bacteria. Hematology evaluation(CBC) is also conducted. CRP analysis is performed on residual plasmacollected from each whole blood sample after processing. CRP analysis isalso conducted on terminal blood samples collected in EDTA tubes if theplasma is able to be isolated. Coagulation assays include prothrombintime (PT), activated partial thromboplastin time (aPTT), fibrinogen, andD-dimer.

Statistical Analysis: Fisher's exact tests are used to compare survivalrates between each antibiotic treatment group and the control group. Atime-to-death analysis is performed on these data determining if thereare differences in protection for the different groups based on a lengthof survival model. The Kaplan-Meier estimates of survival probabilitiesare plotted. The log-rank or Wilcoxon test or Cox proportional hazardregression are used to determine if there are significant differencesbetween the groups, and if so, which groups are significantly different.Bacteremia culture data is analyzed separately at each time point.Summary statistics with 95 percent confidence intervals are produced foreach group and time point. Fisher's exact test is used to test whetherthere are any significant differences in the bacteremia culture databetween groups. For hematology, clinical chemistry, coagulation,temperature, peak and trough antibiotic levels; summary statistics and95% confidence intervals are produced for each group. Analysis ofvariance (ANOVA) models can be fitted to each parameter at each timepoint to determine if there are statistically significant differencesbetween the treatment groups. Few control animals are expected tosurvive, baseline values for treated animals may be used in thesecomparisons, with each animal serving as its own control. Mean changesin temperature, clinical chemistry, and hematology parameters arecompared to zero, to evaluate any change in health status.

Efficacy of CEM-101 against a Lethal Inhalational F. tularensis, Y.pestis, and B. mallei Challenge in NHPs and Pilot Efficacy of CEM-101against a Lethal Inhalational B. anthracis Challenge in NHPs aredetermined using the methods described above.

Example Rabbit Pharmacokinetics and Tolerability Study

TABLE 1 Groups Treatment No. of Animals Dose Level Group Product per Sex(Total) (mg/kg) 1 CEM-101 5 (10) 5 2 CEM-101 5 (10) 10 3 CEM-101 5 (10)15 4 Control 5 (10) NA

TABLE 2 Blood Draw Schedule Antibiotic Clinical Clinical Time PointLevels Chemistry Hematology  Day −7 X X X  Day −3 X X X  *1 hr X X X  *2hr X X X  *4 hr X X X  *6 hr X X X *12 hr X X X *18 hr X X X *24 hr X XX *48 X X X *72 X X X *96 X X X *Day 10 X X X *Day 20 X X X *Day 30 X XX *Blood draws collected post-treatment

Blood levels of CEM-10 are determined from blood samples collected fromtreated and control rabbits at the indicated time before and duringadministration of CEM-101.

Example

Pharmacokinetics (PK) in non-human primates. The PK data is collected inthree phases. Eight monkeys (4 male, 4 female) participate in each phase(Table 3). Each phase is followed by a 7-day minimum washout periodprior to initiation of the next phase.

TABLE 3 Study Design CEM-101 # per Dose Phase Phase (mg/kg) BloodCollection 1 8 5 Day 1: 0^(a), 1, 2, 4, 6, 8, 12, 18, 24, 48 hr, 96 hr 28 10 Day 1: 0^(a), 1, 2, 4, 6, 8, 12, 18, 24, 48 hr, 96 hr 3 8 15 Day 1:0^(a), 1, 2, 4, 6, 8, 12, 18, 24, 48 hr, 96 hr

Example

Characterization of Burkholderia mallei Challenge Material and Deliveryin the Large Animal Exposure system. The strain of Burkholderia malleiis streaked for isolation on appropriate growth media (such as Lysogenybroth (LB) agar with 4 percent glycerol or Ashdown's medium). Uponconfirmation of colony purity and morphology (considering thatmorphologically variant colonies are common for B. mallei), all coloniesare removed and suspended in an appropriate bacterial storage media andfrozen at ≦−70° C. A collection of colonies are prepared rather than asingle isolated colony (it is appreciated that isolating a single colonytype may inadvertently select a variant with altered virulenceproperties). The collected colonies constitute the master cell bank. Theprocedure is repeated by streaking from the master cell bank to preparethe working cell bank.

All cultures prepared on agar media in the preparation of the master andworking cell banks are examined for colony morphology and purity. Grossmorphological examination includes descriptions of colony shape, size,elevation, margin, color, surface appearance, density, and consistency.If a cell bank is determined to contain contaminants, it is re-derivedfrom the master or cell bank.

Culture material from master and working cell banks are Gram-stained.Gram-stain results and gross cellular morphology are observed andcompared to previous results in the published literature. Working cellbank material is used to inoculate broth cultures; flasks are incubatedfor various times and the optical density of the cultures measured.Multiple broth cultures are prepared. Each culture is grown to a targetoptical density and then the numbers of CFU/mL of culture are determinedby spread plate enumeration. A ratio of the number of CFU/mL isdetermined by averaging the results of multiple cultures for one culturedensity. Real-time qualitative PCR (+/−PCR) is performed to determine,at the nucleic acid level, that each working cell bank is indeed the B.mallei strain provided. The concentration of bacteria in the master andworking cell banks for all strains is determined by spread plateenumeration, or comparable technique.

Fresh B. mallei suspensions are prepared from stock samples. A freshbatch is prepared for each day aerosol tests are conducted. Starting(nebulizer) B. mallei solutions for aerosolization diluted in sterilephosphate buffered saline (PBS) containing 0.01% (wt/vol) gelatin with9.7% trehalose (wt/vol) (BSGT) and approximately 8 mL are placed intothe nebulizer for each test. Aerosol samples are collected from theexposure chamber using glass impingers (e.g. model 7541 Ace Glass, Inc.)filled with ˜20 mL of sterile phosphate buffered saline (PBS) containing0.01% (wt/vol) gelatin (BSG) and 0.01% anti-foam A until plating for cfucounts. The aerosol particle size distribution are sampled from theexposure system and measured using an aerodynamic particle sizer.

A series of aerosol exposure system-characterization tests are performedto quantify the aerosol concentration of viable B. mallei (i.e. CFU) andthe reproducibility of aerosol generation. Table 4 illustrates a testmatrix that can be used to test for reproducibility of the aerosolsystem. The characterization tests are repeated to generate fourdifferent, targeted aerosol concentrations on each day for 3 days. Thegeneration of each targeted aerosol concentration is repeated threetimes on each day. Aerosol samples are collected and enumerated for CFUvia the spread plate technique. Additionally, the temperature, relativehumidity, and aerosol particle size during each test is monitored for atleast one time point.

TABLE 4 Aerosol Characterization Testing Target aerosol Number of testiterations per Test number concentration (CFU/L air) target aerosolconcentration Test 1 A 3 Test 2 B 3 Test 3 C 3 Test 4 D 3The complete test matrix (Tests 1-4) is repeated over 3 separate days.

For aerosol characterization testing, statistical ANOVA models arefitted to the test data for the aerosol system. These data consist offour aerosol concentrations tested three times on each day with theentire experiment repeated on three different days. A statisticalhypothesis is tested for whether the measured system output (e.g., sprayfactor) is reproducible (i.e., not statistically significantly differenton one day compared to another). A negative result (i.e., notstatistically significant) will conclude reproducibility. Power analysiswill also be performed to determine the minimum sensitivity of the testto identifying true differences

Example

Development of the mouse aerosol system for generation, delivery, andcollection of B. mallei. A series of aerosol exposuresystem-characterization tests are performed to quantify the aerosolconcentration of viable B. mallei (i.e. CFU) and the reproducibility ofthe exposure system. Table 4 illustrates the proposed tentative testmatrix that is used to test for reproducibility of the aerosol system.The characterization tests are repeated to generate four different,targeted aerosol concentrations on each day for 3 days. The generationof each targeted aerosol concentration is repeated three times on eachday. Aerosol samples are collected as described above and enumerated forcolony forming units (cfu) via a spread plate technique.

For aerosol characterization testing, statistical ANOVA models arefitted to the test data for the aerosol system. As described above, thisdata consists of four aerosol concentrations tested three times on eachday with the entire experiment repeated on three different days. Astatistical hypothesis is tested for whether the measured system output(e.g., spray factor) is reproducible (i.e., not statisticallysignificantly different on one day compared to another). A negativeresult (i.e., not statistically significant) concludes reproducibility.Power analysis is also performed to determine the minimum sensitivity ofthe test to identifying true differences.

Example

Determining the Inhaled Median Lethal Dose (LD₅₀) of B. mallei in Mice.The determination of the inhaled LD₅₀ for B. mallei is conducted inphases. Each phase of this study consists of a post-challenge periodwhich will extend to Day 28. The day an animal is challenged isdesignated as study Day 0. Phase I consists of 5 groups, phase II fourgroups and phases III and IV consist of three groups each. Based on themortality results of the previous phases, new target exposure doses aredetermined. This phased approach allows for increased confidence in theinhaled LD₅₀ value.

TABLE 6 Phase Approach to Determine the LD₅₀ in Mice Phase Groups perPhase Total Mice I 5 groups of 4 20 II 4 groups of 8 32 III 3 groups of8 24 IV 3 groups of 8 24

Mice are challenged via the inhalation route with B. mallei on Study Day0 (for each Phase). A nose-only aerosol exposure system (for example aCH Technologies Tower) is utilized to deliver the desired aerosol dosesto mice. The CH Technologies Tower system used for the mouse aerosolchallenge testing is capable of exposing up to 30 mice at a time withthe addition of impinger samplers, an aerosol particle size analyzer,temperature and humidity monitoring, and mass flow meters (MFM) and massflow controllers (MFC) to control and/or monitor the aerosol flows.Briefly, forced air enters the system through high efficiencyparticulate air (HEPA) filters and is divided into a continuous airstream (continuous air) and an air stream that either flows into theCollison nebulizer (during aerosol generation) or by-passes it (betweenaerosol generations). MFC regulate the flow for each of the air streams.The B. mallei aerosol, created by the nebulizer, is mixed withcontinuous air before delivery to the exposure tower. The aerosolexposure parameters required to deliver these doses are based on resultsfrom the proposed mouse aerosol system characterization study describedabove.

Aerosol concentrations of B. mallei are quantified by determination ofcolony forming units (cfu). Effluent streams are collected directly froman animal exposure port by an in-line all-glass impinger. Serialdilutions of impinger samples are plated and enumerated.

The mice are observed twice daily for a total of 29 days, which includesStudy Day 0 through Study Day 28, to determine the onset of clinicalsigns of disease and survivability.

For LD₅₀ (inhaled median lethal dose) determinations in mice,experiments are conducted in phases (Feder, 1992). Probit dose-responsemodels are fitted to dose-lethality data for mice using the method ofmaximum likelihood (Finney, 1971 and Feder, 1991). Estimated parametersof the probit dose-response models are used to compute the LD₅₀.Fieller's method (Finney, 1971) or other appropriate methods are used tocompute a 95 percent confidence interval for the LD₅₀.

Time to onset of each of the clinical signs are recorded until death,euthanasia, or end of the clinical observation period of the animal. Theproportion of animals showing each of the clinical signs and the time toonset of each sign are determined. Mortality is recorded at the timeobserved. Cox proportional hazards models are fitted to the time toonset and time to death data, with dose as an explanatory variable.Because more severe signs may mask less severe signs, signs may begrouped for this analysis. The median time to signs and median time todeath is calculated at the LD₅₀.

Example

Natural History/LD₅₀ Determination of Inhalational Burkholderia malleiDisease in Cynomolgus macaques. Determination of the lethal dose (LD₅₀)of B. mallei and characterization of the natural history of inhalationalmelioidosis in cynomolgus macaques is determined by monitoring clinicalsigns of disease including clinical observations, hematology, clinicalchemistry, telemetric parameters, coagulation assays, bacteremia, andnumbers of bacteria in selected organs.

Twenty two (11 male, 11 female) naïve cynomolgus macaques (monkeys,NHPs) that are approximately 2.5-5.0 kg (˜2-5 years of age) are obtained(available from Covance). Two of the NHPs will serve as replacements, ifnecessary, during Phases I, II, and III. Monkeys are tested and verifiednegative for tuberculosis and also prescreened within 30 days prior toreceipt to confirm that they are seronegative for SimianImmunodeficiency Virus (SIV), Simian T-Lymphotrophic Virus-1 (STLV-1),and Cercopithecine herpesvirus 1 (Herpes B virus) and negative forSimian Retrovirus (SRV1 & SRV2) by PCR. Monkeys are quarantined for aminimum of five weeks prior to being placed on study. Prior to placementon study all monkeys are surgically implanted with telemetrytransmitters (for example, item D70-PCTP, purchased from Data SciencesInternational, DSI).

Ten groups of monkeys are exposed to various doses of B. malleidelivered via aerosol over a period of four phases (see Table 7). Basedon the mortality results of the previous phases, new target exposuredoses are determined for the succeeding phase. This phased approachallows for increased confidence in the estimated inhaled LD₅₀ value. Thefirst three phases are used to determine the LD₅₀ of B. mallei in NHPs.In the final phase, the remaining 4 NHPs are challenged with a highexposure expected to be lethal in a majority of animals based on theresults from the first three phases (i.e. target an exposure dose aroundthe LD₉₀). If the replacement NHPs are not used in Phases I, II, or III,then they also are challenged in Phase IV. The information collected inPhase IV supports the natural history information collected in the first3 phases and increases understanding of the disease progression inanimals challenged with elevated doses similar to those which might beutilized in efficacy studies. Results obtained from Phase IV may alsoincrease the precision of an LD₉₀ estimate.

TABLE 7 Phase Approach to Determine the LD₅₀ in Cynomolgus MacaquesPhase Group CFU dose Number of NHPs I 1 10,000 2 2 100,000 2 3 1,000,0002 4 10,000,000 2 II 5 TBD 2 6 TBD 2 III 7 TBD 2 8 TBD 2 IV* 9 TBD 2 10TBD 2 *Two additional NHPs are included in this phase if they are notused as replacements in Phases I, II, or III.

The animals are weighed during quarantine and on Study Days −7 and 0.Monkeys which die or are euthanized on study are weighed prior tonecropsy. Additional weights may be taken if an animal appears to belosing weight during the course of disease progression. Clinicalobservations of all monkeys are performed twice daily during thepre-exposure period. Following aerosol exposure, monkeys are monitoredthree times daily for 28 days post-exposure for survival and clinicalsigns of illness: moribund, respiratory distress, appetite, activity(recumbent, weak, or unresponsive), seizures, and other clinicalobservations (described by observer).

Blood samples are taken from a femoral artery or vein, saphenous vein,or other appropriate vein on the days specified in Table 8. Bloodsamples collected on the day of exposure will take place prior toexposure.

Telemetry: Baseline and post-challenge data for body temperature; ECG;activity; and cardiovascular function (heart rate, systolic/diastolicpressure, pulse pressure, mean pressure, and respiratory pressure) arecollected for at least 30 seconds every 15 minutes throughout the study.Baseline data are collected for at least 10 days prior to challenge.Details on the specific operation and data collection are contained inSOP BBRC.VI-087, SOP BBRC.VI-093, and SOP BBRC.VI-096.

TABLE 8 Natural History Blood Draw Schedule Bac- Clinical CoagulationTime Point teremia CBC/CRP Chemistry Assays QPCR Day −7 X X X X X Day 0X X X Day 1 PC X X X X X Day 2 PC X X X X X Day 3 PC X X X X X Day 4 PCX X X X X Day 5 PC X X X X X Day 6 PC X X X X X Day 7 PC X X X X X Day14 PC X X X X X Day 21 PC X X X X X Day 28 PC X X X X X Terminal X CRPonly X

Monkeys are transported into the BL-3˜14 days prior to exposure to allowtime for acclimation. On Day 0, monkeys are anesthetized with Telazol(1-6 mg/kg, IM) and placed into a plethysmography chamber and a ClassIII cabinet system and exposed to the targeted dose of B. malleiaerosolized by a Collison nebulizer and delivered via a head-onlyinhalation exposure chamber. Aerosol concentrations of B. mallei arequantified by determination of CFU. Effluent streams are collecteddirectly from an animal exposure port by an in-line all-glass impinger.Serial dilutions of impinger samples are plated and enumerated.

Monkeys surviving the 28 day post-exposure period are euthanized andexamined as described below. Because of the potential for survivingmonkeys to develop a chronic infection, all animals must be euthanized.In addition, the quantification of bacterial load in the tissues ofanimals in subsequent efficacy studies may provide a secondary endpointfor analysis (i.e. if the treatment doesn't significantly reducemortality it may significantly reduce bacterial load).

Blood samples from time points indicated in Table 8 are cultured todetermine the presence or absence of B. mallei . In addition, at each ofthese time points approximately 100 μl of whole blood is collected fromwhich DNA is isolated and QPCR analysis performed.

Hematology evaluation (CBC) is conducted includes, but is not be limitedto, the following parameters:

White blood cell count (WBC) Mean corpuscular volume (MCV) Differentialleukocyte (% and Mean corpuscular hemoglobin (MCH) absolute) countNeutrophil:Lymphocyte Ratio Mean corpuscular hemoglobin (N/L Ratio)concentration Hemoglobin (HGB) Red cell distribution width (RDW)Hematocrit (HCT) Platelet count (PLT) Red blood cell count (RBC) Meanplatelet volume (MPV)

Clinical chemistry evaluation is conducted andl includes, but is notlimited to, the following parameters:

Alanine aminotransferase (ALT) Albumin/Globulin (A/G) Ratio Aspartateaminotransferase (AST) Blood urea nitrogen (BUN) Alkaline Phosphatase(ALP) Creatinine Gamma-Glutamyl Transferase (GGT) BUN/Creatinine RatioLactate dehydrogenase (LDH) Glucose Sorbitol Dehydrogenase (SDH) SodiumTotal bilirubin Potassium Total protein Chloride Albumin CalciumGlobulin Phosphorus

Blood samples for analysis of coagulation factors are collected intotubes containing sodium citrate. C-Reactive protein (CRP) analysis isperformed on residual plasma collected from each whole blood sampleafter processing. CRP analysis is also conducted on terminal bloodsamples collected in EDTA tubes if the plasma can be isolated.

Gross necropsy is performed on all monkeys that die or are euthanized.Portions of target tissues including but not limited to lungs, spleen,and liver are homogenized, the DNA isolated, and qPCR performed todetermine bacterial loads in these organs. Sections of target tissuesincluding but not limited to brain, lungs, kidney, liver, spleen,mediastinal and bronchial lymph nodes as well as all gross lesions arepreserved in 10% neutral buffered formalin. Histopathology is performedon all animals including survivors euthanized on study day 28.

Probit dose-response models are fitted to dose-lethality data formonkeys using the method of maximum likelihood (Finney, 1971 and Feder,1991). Estimated parameters of the probit dose-response models are usedto compute the LD₅₀ and LD₉₀. Fieller's method (Finney, 1971) or otherappropriate methods are used to compute a 95 percent confidence intervalfor the LD₅₀ and LD₉₀.

Time to onset of each of the clinical signs is recorded until death,euthanasia, or end of the clinical observation period of the animal. Theproportion of animals showing each of the clinical signs and the time toonset of each sign is determined. Cox proportional hazards models arefitted to the time to onset and time to death data, with dose as anexplanatory variable. Because more severe signs may mask less severesigns, signs may be grouped for this analysis. The median time to signsof illness and median time to death are calculated at the LD₅₀ dose.

Statistical Analysis of Hematology, Clinical Chemistry, Coagulation andqPCR results: These parameters are evaluated relative to baseline forthe same animal by calculating the shift from the mean pre-challengelevel at each data collection time point. If necessary, hematology andclinical chemistry data are first transformed by the logarithm toproduce a more normally distributed response. In this case, finalresults are returned to their original units through exponentiation.Summary statistics are provided for the data. At each time point, themean shift from baseline for all animals is evaluated using t-tests todetermine if the parameter shift is statistically significantlydifferent than no shift. For each of the telemetry parameters, datacollected immediately prior to challenge are averaged for each animal,resulting in baseline averages. Telemetry results in the post-challengeperiod are then adjusted for each animal by subtracting the baselineaverage. Further data smoothing may be employed by moving averages orother appropriate method. The time trend in the baseline adjusted andsmoothed data measurements as well as the cumulative sum of adjustmentsfor each telemetry parameter are plotted for all animals.

Specific criteria are established for onset of disease using thetelemetry data. Statistical tests are determined to assess the specifictime point for which a disease criterion occurs in some proportion ofthe population and/or provide a time interval around a certain meanmanifestation of disease. Where possible and judged relevant, theadditional data of time to death, hematology, bacteriology, and clinicalobservations may also be incorporated into this analysis to determinethe optimum timing of initiation of therapy.

Example

Efficacy of CEM-101 Against a Lethal Inhalational F. Tularensis, Y.Pestis, and B. Mallei Challenge in BALB/c Mice

TABLE 9 Study Design CEM-101 Dose Number Treatment Duration Group(mg/kg) of mice (days) 1 50 20 14 2 100 20 14 3 150 20 14 4 200 20 14 5Control 20 14

One hundred and ten (110) BALB/c mice are used in this procedure (100 tobe placed on study and 10 extras). On day 0, mice are placed into anose-only aerosol exposure system and exposed to agent aerosolized by aCollison nebulizer. Aerosol concentrations of agent are quantified bydetermination of colony forming units (cfu). Effluent streams arecollected directly from an exposure port by an in-line all-glassimpinger. Serial dilutions of impinger samples are plated andenumerated.

Following challenge, mice are observed twice daily for 28 days forsurvival and clinical signs of illness. The bacterial burden in thelungs, spleen and peripheral blood are determined by quantitative PCRfrom all mice found dead or euthanized. All animals surviving the 28 daypost-challenge observation period are anesthetized, and a terminal bloodsample is collected followed by immediate euthanasia. Followingeuthanasia a specimen of lung and spleen are also collected forbacterial burden analysis.

Fisher's exact tests are used to compare survival rates between eachantibiotic treatment group and the control group. A time-to-deathanalysis are performed on these data to determine if there aredifferences in protection for the different groups based on a length ofsurvival model.

Pilot Efficacy and Pivotal Efficacy of CEM-101 against a LethalInhalational B. anthracis Challenge in Rabbits (see general Rabbitefficacy study outline below).

Using the methods described above the efficacy of CEM-101 againstinhalation challenge of rabbits with aerosolized of B. anthracis

TABLE 12 Rabbit Post-Exposure Efficacy Studies (used for B. anthracis).Hematology/Clinical CEM-101 Treatment Chemistry/Bacteremia Dose Numberof Duration Analysis (relative Group (mg/kg) animals (days) to the dayof challenge) 1 5 10 14 −7, 1, 2, 3, 4, 7, 14, 21, 28 2 10 10 14 −7, 1,2, 3, 4, 7, 14, 21, 28 3 15 10 14 −7, 1, 2, 3, 4, 7, 14, 21, 28 4Control 10 14 −7, 1, 2, 3, 4, 7, 14, 21, 28

Animals are treated between 18-24 hours post challenge. All animals aremonitored for 28 days post challenge. Blood draws for CEM-101 peak andtrough levels are collected after first, middle and last antibiotictreatment.

Example

MICs were determined by the microdilution method in 96-well platesaccording to Clinical and Laboratory Standards Institute (CLSI formallyNCCLS) (1). Antibiotics were serially diluted twofold in 50 μl ofcation-adjusted Mueller-Hinton broth (CAMHB). For F. tularensisdeterminations CAMHB was supplemented with 2% Isovitalex (BectonDickinson). The antibiotic range was 16 to 0.008 μg/ml based on a finalwell volume of 100 μl after inoculation.

Inocula were prepared by suspending colonies from a 18-24 h (B.anthracis, B. mallei or B. pseudomallei) or 48 hr (Y. pestis, F.tularensis) sheep blood (SBA) or chocolate agar plate (according toCLSI). Suspended cultures were diluted with CAMHB to a bacterial celldensity of 10⁶ CFU/ml. To each well of the 96-well plate, 50 μl of thisdilution was added for a final inoculum of approximately 5×10⁴ CFU/well.Plates were incubated at 35° C. MICs were determined visually at 24- and48 h and by reading the plates at 600 nm (SpectroMax M2, MolecularDevices).

Inoculum preparation and antibiotic microdilution were performedaccording to CLSI methods. MICs of each agent were determined by themicrodilution method in 96-well plates, after an 18- or 42-h incubationat 35° C., for 30 strains representing the genetic and geographicdiversity of each bacterial species.

Quality control of antibiotic stocks was established by usingStaphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922.

# Range MIC50 MIC90 Strains ug/ml ug/ml ug/ml B. anthracis 30<0.008-0.015 <0.008 <0.008 Y. pestis 30 0.25-2   1 2 F. tularensis 30<0.08-4   0.03 2 B. mallei 30 0.25-2   1 1 B. pseudomallei 30 16 16 16Antibiotic susceptibility testing indicated that all of bacterialspecies in this study with the exception of B. pseudomallei are intherapeutically achievable ranges for CEM-101. B. pseudomallei has ademonstrated multi-drug efflux system that is active againstmacrolides(2) and the data indicating CEM-101 resistance are consistent.The F. tularensis distribution reflects overlap of the two distinctbiovars “A” and “B” of this species. The B strains distribute to thehigher MICs while the A strains distribute to the lower end. A strainsare the more virulent and pose the greater BW/BT threat making theirgreater susceptibility fortuitous.

Since many of these bacterial agents are intracellular during infectionthe observed ability of many macrolides to preferentially accumulate incells, may enhance efficacy among individuals exposed to aerosolizedBW/BT agents. The demonstrated broad-spectrum activity against a varietyof potential BW/BT bacterial agents along with oral bioavailabilitymakes CEM-101 an attractive candidate for treatment after exposures andinfections. Efficacy of CEM-101 in the animal-infection models for theseagents should be evaluated.

Example

Human THP-1 macrophages were used. Accumulation was measured bymicrobiological assay. Intracellular activity was determined againstphagocytized S. aureus (ATCC 25923; MICs: CEM-101, 0.125 mg/L; AZI, 0.5mg/L) using a dose-response approach (AAC 2006; 50:841-51). Verapamil(100 μM) and gemfibrozil (250 μM) were used as inhibitors ofP-glycoprotein and MRP, respectively (AAC, 2007; 51:2748-57).

Accumulations and activities after 24 h incubation, with and withoutefflux transporters inhibitors, are shown in the following Table, whereCc/Ce is the apparent cellular to extracellular concentration ratio, andE_(max) is the maximal decrease of intracellular cfu compared topost-phagocytosis inoculum (calculated from non-linear regression[sigmoidal] of dose-effect response experiments).

AZI CEM-101 Intracellular activity Intracellular activity (Δ log cfu at24 h) (Δ log cfu at 24 h) Cc/Ce ¹ Static dose Cc/Ce ¹ Static doseCondition (24 h) (mg/L) E_(max) ² (24 h) (mg/L) E_(max) ² control  127.7± 23.5 ~7.0  0.10 ± 0.09 268.1 ± 7.1 ~0.02 −0.85 ± 0.23 ^((b)) Verapamil216.37 ± 46.6 ^((a)) ~0.2 −0.37 ± 0.15 290.2 ± 12.9 ~0.03 −0.59 ± 0.22^((b)) Gemfibrozil 129.12 ± 2.69 ~3.8 −0.12 ± 0.20 308.2 ± 47.8 ~0.03−0.73 ± 0.20 ^((b)) ^((a)) Statistically significant from both controland Gemfibrozil; ^((b)) not statistically significant.

Example

Intracellular activity of antibiotics. The determination of antibioticactivity against intraphagocytic S. aureus strain ATCC 25923 wasdetermined. Full dose-responses studies were performed to assess theimpact of active efflux in the modulation of the intracellular activityof CEM-101 and AZI against intraphagocytic S. aureus (strain ATCC 25923[MICs: CEM-101, 0.125mg/L; AZI, 0.5 mg/L]. Antibiotics were compared at24h for: (i) their relative static concentration (Cs), and (ii) theirrelative maximal efficacy (E). While verapamil (but not gemfibrozil)increases the intracellular activity of AZI, neither inhibitor havesignificant effect on the activity of CEM-101, suggesting that thelatter, in contrast with AZI, is not a substrate of the correspondingeukaryotic transporters.

Example

Cellular accumulation of antibiotics. The cellular content in macrolideswas measured in THP-1 macrophages by microbiological assay, using S.aureus ATCC 25923 as test organism. Cell proteins was assayed inparallel using the Folin-Ciocalteu/Biuret method. The cell associatedcontent in macrolides was expressed by reference to the total cellprotein content, and converted into apparent concentrations using aconversion factor of 5 μL per mg of cell protein (as commonly used forcultured cells).

The cellular accumulation of CEM-101 in comparison with that of AZI inTHP-1 cells was first measured FIG. 7 (panel A). At 24 h, bothantibiotics concentrate to large extents in cells, but with a largervalue (Cc/Ce) for CEM-101. In a second stage, whether CEM-101 is asubstrate of Pgp or MRP efflux transporters was investigated FIG. 7(panel B). Using a Pgp (verapamil) or MRPs inhibitor (gemfibrozil), nosignificant variations of the cellular accumulation of CEM-101 areobserved while verapamil increases significantly the cellularaccumulation of AZI.

Uptake of CEM-101 was linear over time, reaching accumulation levelsabout 375-fold within 24 h (AZI, 160X, CLR, 30X, TEL, 21X). Accumulationwas suppressed by acid pH or addition of the proton ionophore monensin,but not modified by verapamil or gemfibrozil (preferential inhibitors ofPgp and MRP, respectively). Panel B shows that the accumulation of bothCEM-101 and AZI was reduced when the experiments were conducted atacidic pH, with the change occurring almost entirely when the pH wasbrought from 7 to 6. Panel C shows that monensin, which is known todecrease the cellular accumulation of many weak organic bases, alsoalmost completely suppressed the accumulation of both CEM-101 and AZI.In contrast, verapamil, an inhibitor of the P-glycoprotein effluxtransporter (Pgp, also known as MDR1), increased the accumulation of AZIwithout affecting that of CEM-101, whereas gemfibrozil, an inhibitor ofmultidrug resistance proteins (MRP) and other organic anion transportersdid not affect either compound. Neither verapamil nor gemfibrozilaffected the accumulation of TEL or CLR (data not shown). The efflux ofCEM-101 from cells incubated with 10 mg/L of CEM-101 for 1 h and thentransferred into drug-free medium was examined. Efflux proceeded in abimodal fashion, with half of the cell-associated drug being releasedwithin approximately 10 min, followed by a slower release phase ofseveral hours (data not shown).

Example

Macrolides accumulate in eukaryotic cells and are consideredadvantageous for the treatment of intracellular infections. Ketolidesare active against erythromycin-resistant organisms. The cellularaccumulation and intracellular activity of CEM-101 towards theintracellular forms of Staphylococcus aureus (S. a.), Listeriamonocytogenes (L. m.), and Legionella pneumophila (L. p.) in comparisonwith AZI, CLR, and TEL is shown in the following table.

MIC^(a) Cs^(b) E_(max) ^(c) CEM-101 S. a. 0.06 0.022 −0.86 L. m. 0.0040.11 −0.66 L. p. 0.004 0.018 −1.03 AZI S. a. 0.5 >50 0.04 L. m. 1 11.6−0.81 L. p. 0.016 2.90 −0.83 CLR S. a. 0.5 0.84 −0.18 L. m. L. p. 0.0070.12 −0.71 TEL S. a. 0.25 0.63 −0.29 L. m. L. p. 0.007 0.06 −0.63^(a)mg/L; ^(b)static concentration (mg/L) at 24 h; ^(c)Δ log₁₀ CFU at 24h compared to the post-phagocytosis inoculum

Example

MICs and extracellular activities of antibiotics were determined in MHBat both neutral and acidic pH. Intracellular activity was determinedagainst S. aureus (ATCC 25923) phagocytosed by THP-1 macrophages aspreviously described (AAC, 2006, 50:841-851). Results were expressed asa change of efficacy compared to time 0 h.

Conditions CEM-101 AZI CLR TEL MICs (mg/L) (i) pH 7.4 0.125 0.5 0.5 0.5(ii) pH 5.5 1-2 256 16 8 Extracellular activity (24 h): Δ log cfu fromtime 0 h (i) Broth pH 7.4 Emax¹ −1.4 ± 0.1 −1.2 ± 0.6 −1.4 ± 0.2 −1.0 ±0.4 Static ~0.06 ~3.63 ~1.41 ~0.28 dose² R² 0.964 0.860 0.965 0.868 (ii)Broth pH 5.5 Emax¹ −1.6 ± 0.4 +2.1 ± 0.1 −1.5 ± 0.8 −1.4 ± 0.9 Static~1.48 / ~10.47 ~9.33 dose² R² 0.915 / 0.911 0.879 Intracellular activity(24 h): Δ log cfu from time 0 h Emax¹ −0.8 ± 0.2 0.10 ± 0.0 −0.1 ± 0.1−0.4 ± 0.2 Static ~0.02 ~7.8 ~0.98 ~0.23 dose² R² 0.906 0.980 0.9740.935 THP-1 Emax¹ −0.8 ± 0.2  0.1 ± 0.1 −0.1 ± 0.1 −0.4 ± 0.1 Static~0.02 ~10 ~0.98 ~0.28 dose² ¹Maximal decrease of intracellular cfucompared to initial, post-phagocytosis inoculum (calculated fromnon-linear regression [sigmoidal] of dose-effect response) run in broth(extracell.) or with infected macrophages (intracell.) ²Extracellularconcentration (Cs in mg/L) yielding an apparent static effect.Comparative pharmacological descriptors (Emax and static concentrations[Cs]) obtained from the dose-responses studies. Dose-response studies inMueller-Hinton broth. Against S. aureus ATCC 25923 and in broth, at pH7.4, CEM-101 is systematically more active than AZI, CLR and TEL; at pH5.5, AZI, CLR and TEL show significant decrease of their potencies,while CEM-101 shows less change.Compared to AZI, CLR and TEL, CEM-101 activity was less affected byacidic pH of the broth and showed greater potency (lower static dose)and larger maximal efficacy (Emax) against intracellular S. aureus.

Example

Cell lines. Experiments were performed with THP-1 cells (ATCC TIB-202;American Tissue Culture Collection, Manassas, Va.), a humanmyelomonocytic cell line displaying macrophage-like activity (see, e.g.,Barcia-Macay et al., Antimicrob. Agents Chemother. 50:841-851 (2006)).Assay of the cell-associated macrolides and calculation of the apparentcellular- to-extracellular-concentration ratios. Macrolides were assayedby a microbiological method, using S. aureus ATCC 25923 as a testorganism. Cell proteins were measured in parallel using theFolin-Ciocalteu/biuret method. The cell-associated contents inmacrolides were expressed by reference to the total cell protein contentand converted into apparent concentrations using a conversion factor of5 μL per mg of cell protein, an average value found for many culturedcells.

Bacterial strains, susceptibility testing, and 24-h dose-response curvestudies with broth. S. aureus ATCC 25923 (methicillin [meticillin]sensitive), L. monocytogenes strain EGD, and L. pneumophila strain ATCC33153 were used in the present study. MIC determinations were performedin Mueller-Hinton broth (for S. aureus) and tryptic soy broth (for L.monocytogenes) after a 24-h incubation, or in α-ketoglutarate-bufferedyeast extract broth (for L. pneumophila) after a 48-h incubation. For S.aureus studies, 24-h concentration-response experiments in acellularmedium were performed in Mueller-Hinton broth.

Cell infection and assessment of antibiotic intracellular activities.Infection of THP-1 cells and assessment of the intracellular activity ofantibiotics were performed using conventional methods for S. aureus andL. monocytogenes or with minor adaptations for L. pneumophila using (i)a multiplicity of infection of 10 bacteria per macrophage and (ii)gentamicin (50 mg/liter) for 30 to 45 min for the elimination ofnonphagocytosed bacteria.

Statistical analyses. Curve-fitting statistical analyses were performedwith GraphPad Prism version 4.03 and GraphPad Instat version 3.06(GraphPad Software, San Diego, Calif.).

Example

Susceptibility toward S. aureus ATCC 25923, Listeria monocytogenes EGD,and Legionella pneumophila ATCC 33153. CEM-101 showed lower MICs thanAZI against the three selected organisms (S. aureus, 0.06 and 0.5mg/liter; L. monocytogenes, 0.004 and 1 mg/liter; and L. pneumophila,0.004 and 0.016 mg/liter) in conventional susceptibility testing. TheMICs of CEM-101, TEL, AZI, and CLR against S. aureus and L.monocytogenes were measured in broths adjusted to pH values ranging from5.5 to 7.4. The range was selected to cover the values at which theantibiotics could be exposed in the extracellular milieu orintracellularly for the two organisms considered. As illustrated in FIG.3, all four drugs showed a marked decrease in potency against bothorganisms when the pH was decreased from 7.4 to 5.5, with AZIdemonstrating the most significant loss of activity. CEM-101 retainedthe most activity, consistently showing the lowest MICs throughout theentire pH range investigated, with values (mg/liter) ranging from 0.06(pH 7.4) to 0.5 (pH 5.5) for S. aureus (ATCC 25923) and 0.0039 (pH 7.4)to 0.25 (pH 5.5) for L. monocytogenes (EDG). For L. pneumophila (datanot shown), the MIC of CEM-101 increased from 0.005 to 0.01 and that ofAZI from approximately 0.01 to 0.25 mg/liter when the pH of the brothwas decreased from 7.4 to 6.5 (no determination could be made at lowerpH values because of absence of growth).

Example

Time and concentration effects against extracellular and intraphagocyticS. aureus. Short-term (6-h) time-kill curves were obtained for CEM-101in comparison with those for AZI against S. aureus (ATCC 25923) in brothand after phagocytosis by THP-1 macrophages using two single fixedconcentrations of 0.7 and 4 mg/liter. The lower concentration was chosento be relevant to the serum concentration of AZI and CEM-101, and thehigher concentration was selected to be above the MIC of AZI for theorganisms of interest. Results presented in FIG. 5 show that under theseconditions, only CEM-101 was able to significantly decrease CFU in brothas well as in THP-1 macrophages at the 0.7-mg/liter concentration. Atthe 4-mg/liter concentration in broth, AZI eventually achieved the sameantibacterial effect as CEM-101, but at a lower rate (5 h compared to 1h). In THP-1 macrophages, no consistent activity was detected for AZI,even at the 4-mg/liter concentration, whereas CEM-101 again achieved areduction of approximately 1.5 log10 CFU, similar to the magnitude seenat the 0.7-mg/liter concentration. In all situations with CEM-101, themaximal decrease of CFU was obtained within 1 h and was maintainedthereafter.

We then performed concentration-response experiments at a fixed timepoint (24 h) to obtain the pertinent pharmacological descriptors ofCEM-101 activity (relative potency [50% effective concentration {EC50}],apparent static concentration [C_(s)], and relative maximal efficacy[E_(max)] in comparison with CLR, AZI and TEL activity (additionaldetails are described in Barcia-Macay et al., Pharmacodynamic evaluationof the intracellular activities of antibiotics against Staphylococcusaureus in a model of THP-1 macrophages Antimicrob. Agents Chemother.50:841-851 (2006)). Data are presented in FIG. 4 as a function of (i)weight concentrations (mg/liter) and (ii) multiples of the MICs (asdetermined in broth at pH 7.4). The numerical values of thecorresponding pharmacological descriptors are shown in the Table.Pertinent regression parameters^(a) (with confidence intervals [CI]),and statistical analysis of the dose-response curves illustrated in FIG.4.

broth⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄) (CI) C_(S) ^(⋄⋄) R²CEM-101 −1.37 mg/L 0.03 0.06 0.973 (−1.67 to −1.08) (0.02 to 0.06) a; Aa; A x MIC 0.48 0.88 (0.26 to 0.91) a; A TEL −1.00 mg/L 0.12 0.29 0.892(−1.78 to −0.22) (0.03 to 0.52) a; A b; A x MIC 0.46 0.96 (0.11 to 2.06)a; A AZI −1.23 mg/L 1.78 3.4 0.872 (−2.55 to 0.083) (0.45 to 7.02) a; Ac; A x MIC 3.55 6.87 (0.90 to 14.0) b; A CLR −1.41 mg/L 0.80 1.32 0.956(−1.95 to −0.87) (0.41 to 1.56) a; A c; A x MIC 1.59 2.65 (0.81 to 3.1) a, b; A THP-1 macrophages⁺⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄) (CI)C₅ ^(⋄⋄) R² (CI) CEM-101 −0.86 mg/L  0.0068 0.022 0.927  (−1.36 to−0.37) (0.0023 to 0.020) a; B a; B x MIC 0.11  0.35 (0.037 to 0.32) a; BTEL −0.29 mg/L 0.024 0.63 0.954 (−0.70 to 0.12)  (0.007 to 0.088) b; Bb; B x MIC 0.097 1.04 0.027 to 0.35 a; B AZI  0.04 mg/L 0.11  >50 0.983(−0.23 to 0.32)  (0.05 to 0.22) b; B c; B x MIC 0.22  >100  0.11 to 0.45a; B CLR −0.18 mg/L 0.046 0.84 0.974 (−0.52 to 0.16) (0.018 to 0.12) b;B b, c; B x MIC 0.093 1.68 0.035 to 0.25 a; B ^(a)using all data pointsshown in FIG. 6 (data from samples without antibiotic when theextracellular concentration of an antibiotic is lower than 0.01 x MIC(5) ⁺original inoculum [time = 0 h]: 0.97 ± 0.24 × 10⁶ CFU/mL (n = 3)⁺⁺original (post-phagocytosis) inoculum [time = 0 h]: 2.74 ± 0.55 × 10⁶CFU/mg protein (n = 3) ^(♦)CFU decrease (in log₁₀ units) at time = 24 hfrom the corresponding original inoculum, as extrapolated for antibioticconcentration = ∞; samples yielding less than 5 counts were consideredbelow detection level. ^(⋄)concentration (in mg/L or in x MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1); ^(⋄⋄)concentration (in mg/L or in x MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation; StatisticalAnalyses. Analysis of the differences between antibiotics (per columnfor the corresponding rows; one-way ANOVA with Tuckey test for multiplecomparisons between each parameter for all drugs): figures withdifferent lower case letters are significantly different from each other(p < 0.05). Analysis of the differences between broth and THP-1macrophages (per row for the corresponding columns; unpaired, two-tailedt-test): figures with different upper case letters are significantlydifferent from each other (p < 0.05).

The activities in both broth and THP-1 macrophages developed in aconcentration-dependent fashion, as denoted by the sigmoidal shape ofeach best-fit function (Hill equation). In broth, the relative efficacyof CEM-101 (E_(max) of −1.37 log₁₀) was similar to that of the otherdrugs (E_(max) values of −1.00 to −1.41 log₁₀). In THP-1 macrophages,the relative efficacy of CEM-101 was significantly decreased compared tothat in broth (E_(max) of −0.86 log₁₀), but not to the same extent asthose of the other drugs, which essentially became bacteriostatic only(E_(max) values of 0.04 to −0.29 log₁₀). On a weight basis, CEM-101 hadhigher relative potencies (lower E₅₀ values) and lower staticconcentrations (lower C_(s) values) than all three comparator drugs inboth broth and in THP-1 macrophages. When the data were analyzed as afunction of equipotent concentration (multiples of the MIC), thesedifferences in EC₅₀ values were reduced, indicating that the MIC was themain driving parameter in this context. In broth, even when analyzed asmultiples of the MIC, CEM-101 and CLR still showed significantly lowerEC₅₀s than TEL and AZI.

Example

Activity against intraphagoctic L. monocytogenes and L. pneumophila. Thesame approach was used as that for S. aureus to assess the activities ofCEM-101 and AZI against phagocytized L. monocytogenes and L. pneumophilato obtain information on concentration-effect relationships and on thecorresponding pertinent pharmacological descriptors. As shown in FIG. 6,a relationship compatible with the Hill equation was observed in allcases, although the limited growth of L. pneumophila made the fitting offunctions somewhat more uncertain. When the data were plotted againstweight concentration, it appeared that CEM-101 had a higher relativepotency (lower EC50) than AZI for both L. monocytogenes and L.pneumophila. This difference was reduced but nevertheless remainedsignificant when data for L. pneumophila were plotted against multiplesof the MIC, indicating that the MIC was an important but not theexclusive driver of intracellular activity against this organism.Conversely, no difference in the responses was seen for L. monocytogeneswhen data were expressed as multiples of the MIC. Numerical values ofthe pertinent pharmacological descriptors and statistical analysis oftheir differences are shown in the Table.

Pertinent regression parameters^(a) (with confidence intervals [CI]),and statistical analysis of the dose-response curves illustrated in FIG.6.

L. monocytogenes EGD⁺ antibiotic E^(max♦) (CI) EC₅₀ ^(⋄) (CI) C_(S)^(⋄⋄) R² −0.66 mg/L  0.020 0.11 0.934 CEM-101 (−1.28 to −0.037) (0.005to 0.073) a A X MIC 5.00 0.88 (1.36 to 18.5) A AZI −0.81 mg/L 2.66 11.60.953 (−2.11 to 0.48)  (0.91 to 7.73) a B X MIC 2.66 11.6 (0.81 to 3.1) A L. pneumophila ATCC 33153⁺⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄)(CI) C_(S) ^(⋄⋄) R² −1.03 mg/L  0.052 0.018 0.920 CEM-101 (−1.34 to−0.72) (0.012 to 0.23)  a A x MIC 13.1  4.56 (3.02 to 57.0) A AZI −0.83mg/L 2.86 2.90 0.903 (−2.00 to 0.34)  (0.17 to 48.6) a B x MIC 179.0  181  (10.5 to 3038) B ^(a)using all data points shown in FIG. 6 (datafrom samples without antibiotics were not used because of evidence ofextracellular growth when the extracellular concentration of anantibiotic is lower than 0.01 x MIC (5). ⁺original (post-phagocytosis)inoculum [time = O h; CFU/mg protein]): L. monocytogenes, 1.67 ± 0.22 ×10⁶ (n = 3); L. pneumophila, 0.94 ± 0.60 × 10⁶. ^(♦)CFU decrease (inlogo units) at time = 24 h (L. monocytogenes) or 48 h (L. pneumophila)from the corresponding original inoculum, as extrapolated for antibioticconcentration ∞; samples yielding less than 5 counts were consideredbelow detection level. ^(⋄)concentration (in mg/L or in x MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1). ^(⋄⋄)concentration (in mg/L or in x MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation. Statisticalanalyses: analysis of the differences between the two antibiotics (percolumn for the corresponding rows; unpaired, two-tailed t-test): figureswith different lower case letters are significantly different from eachother (p < 0.05).

Example

Dose-response studies in infected THP-1 macrophages Againstintraphagocytic S. aureus ATCC 25923, CEM-101 is more potent than AZI,CLR and TEL (lower Cs), In addition, CEM-101 is able to reduce theintracellular inoculum (E_(max)˜1 log), which is not observed with anyof AZI, CLR and TEL.

CEM-101 Uptake within Cells (ii): Role of the Cell Type

MDCK sur- THP-1 J774 MDCK expressing the (human (murine (canine MDR1efflux Cells macrophages) macrophages) epith. cells) transporters Cc/Ce~50-150 ~60 ~45 ~30 at 5 h

Example

Example Dose-response studies of CEM-101 vs. comparators (AZI, CLR andTEL) against intracellular S. aureus ATCC 25923 (THP-1 macrophages). SeeFIG. 9 and the Table.

CEM-101 AZI CLR TEL Emax −0.80 ± 0.11 0.04 ± 0.11 −0.18 ± 0.13 −0.29 ±0.16 Cs (mg/L) ~0.01 >50 ~0.86 ~0.27

Example

Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages were measured. See FIG. 8 bars represent theMICs (in mg/L) or the extracellular static dose.

METHOD. Mouse peritoneal macrophages were infected with viable M.leprae, the drugs are added and incubated at 33° C. for 3 days. After 3days macrophages were lysed to release the intracellular M. leprae whichwere then assayed for viability by radiorespirometry and viabilitystaining. CEM-101 shows efficacy against intracellular M. lepraeviability.

The Thai-53 isolate of M. leprae, maintained by serial passages inathymic nu/nu mice footpads, was used for all experiments. For axenictesting freshly harvested viable M. leprae were incubated in mediumalong with different concentrations of the drugs (CEM-101, CLR andrifampin) for 7 days at 33° C. At the end of this incubationdrug-treated M. leprae were subjected to radiorespirometry to assessviability based on oxidation of palmitate and staining with viabilitydyes to assess the extent of membrane damage. For intracellular testingperitoneal macrophages from Swiss mice were infected with freshlyharvested viable M. leprae at an MOI of 20:1 for 12 hours. At the end ofthe infection extracellular bacteria were washed and drugs added atdifferent concentrations and incubated for 3 days at 33° C. At the endof 3 days cells were lysed to obtain the intracellular M. leprae forradiorespirometry and viability staining.

CEM-101 at 0.15 μg/ml was able to significantly (P<0.001) reduce theviability of M. leprae in both axenic and intracellular cultures whencompared to controls. Inhibition by CEM-101 was not statisticallydifferent from inhibition obtained with CLR under identical conditionsand at the same concentration.

Example

The high potency of CEM-101 against Streptococcus pneumoniae,β-haemolytic and viridans group streptococci, Staphylococcus spp. andenterococci has been documented in early screening studies performedusing reference Clinical and Laboratory Standards Institute (CLSI)methods. Since mechanisms and occurrences of resistance are increasingrapidly that may compromise the MLSB-ketolide class, the bactericidalactivity (MBC and killing curves) of CEM-101 with five selected classesof antimicrobial agents when testing wild type (WT) andphenotypically/genotypically defined resistant organism subsets wasassessed. MBC determinations for CEM-101, TEL, and CLR used CLSI methodsfor 40 strains (6 species groups). KC used 8 strains (6 species groups).PAE was tested (5 strains) at 4× concentration for 1 or 2 hoursexposure; TEL control.

MBC and killing curve studies: A total of 40 strains (10 S. pneumoniae,10 S. aureus, and 5 each of β-haemolytic streptococci, viridans groupstreptococci, coagulase-negative staphylococci [CoNS] and enterococci)were MIC tested followed by MBC determinations using CLSI procedures(MIC and MBC range, 0.008-16 μg/ml). The lowest concentration of atested agent that killed ≧99.9% of the initial inoculum was defined asthe MBC endpoint (Tables 2 and 3). Time kill bactericidal activity wasperformed for CEM-101, TEL, CLR, and AZI on eight selected strainsaccording to methods described by Moody & Knapp, NCCLS M21-A3 and M26-A.The compounds were tested at 2×, 4×, 8× MIC; and colony counts wereperformed at T0, T2, T4, T8 and T24.

CEM-101 exhibited low MBC/MIC ratios (≦4) for BSA, SA andcoagulase-negative staphylococci; and 2-fold greater potency than TEL.SA, enterococci and some macrolide/ CLN-resistant (R) strains had higherratios. KC results showed more rapid and greater cidal activity(concentration dependant) for CEM-101 compared to TEL. CEM-101 exhibitedcidal activity against several Gram-positive species at rates and anextent greater than TEL.

Distribution of isolates according to MBC/MIC ratio for CEM-101, TEL,CLR and AZI

Organism/Antimicrobial No. of strains with MBC/MIC value of: agent (no.tested) 1 2 4 8 16 ≧32 S. pneumoniae (10) CEM-101 3 5 0 0 0 2Telithromycin 2  6^(a) 0 0 0 2 Clarithromycin 2 3 1 0 0 —^(b)Azithromycin 2 4 0 0 0 —^(b) β-haemolytic streptococci (5) CEM-101 0 1 20 0 2 Telithromycin 0 1 1 1 0 2 Clarithromycin 0 0 1 1 0  2^(b)Azithromycin 0 0 0 0 2  2^(b) Viridans group streptococci (5) CEM-101 30 1 0 0 1 Telithromycin 2 1 1 0 0 1 Clarithromycin 0 0 1 0 0  3^(b)Azithromycin 0 0 0 0 1  3^(b) S. aureus (10) CEM-101 1 0 0 0 1 8Telithromycin 0 0 0 0 0 10  Clarithromycin 0 0 0 0 0  6^(b) Azithromycin0 0 0 0 0  6^(b) Coagulase-neg. staphylococci (5) CEM-101 1 1 0 3 0 0Telithromycin 0 0 0 0 2 3 Clarithromycin 0 0 0 0 0  4^(b) Azithromycin 00 0 0 0  4^(b) Enterococcus spp. (5) CEM-101 0 0 0 0 0 5 Telithromycin 00 0 0 0 5 Clarithromycin 0 0 0 0 0  2^(b) Azithromycin 0 0 0 0 0  2^(b)^(a)Includes six isolates with a MIC of ≦0.008 μg/ml and a MBC of 0.015μg/ml (off scale comparisons). ^(b)MBC was not evaluated on isolateswith resistant level MIC results.

CEM-101 showed rapid bactericidal activity (reduction of ≧3 log10CFU/ml) against macrolide-susceptible strains of S. aureus, S.epidermidis, S. pneumoniae, S. pyogenes (only at 8× MIC) and viridansgroup streptococci, as well as a macrolide-resistant S. pyogenes.CEM-101 produced a greater reduction of CFU/ml and more rapid killingwhen compared to either TEL or the macrolides CLR and AZI.

Summary of time kill curve results. Organism Antimicrobial agentAntimicrobial activity S. aureus CEM-101 Cidal at 2X, 4X, 8X (ATCC29213) Telithromycin Cidal at 8X only Clarithromycin Cidal at 8X onlyAzithromycin Cidal at 8X only S. epidermidis CEM-101 Cidal at 2X, 4X, 8X(095-2777A) Telithromycin Static Clarithromycin Static AzithromycinStatic E. faecalis CEM-101 Static (ATCC 29212) Telithromycin StaticClarithromycin Static Azithromycin Static S. pneumoniae CEM-101 Cidal at2X, 4X, 8X (ATCC 49619) Telithromycin Cidal at 2X, 4X, 8X ClarithromycinCidal at 2X, 4X, 8X (slow killing) Azithromycin Cidal at 2X, 4X, 8X(slow killing) S. pneumoniae CEM-101 Static (075-241B) TelithromycinStatic S. pyogenes CEM-101 Cidal at 8X only (117-1612A) TelithromycinCidal at 8X only (slow killing) Clarithromycin Cidal at 8X only (slowkilling) Azithromycin Cidal at 8X only (slow killing) S. pyogenesCEM-101 Cidal at 2X, 4X, 8X (088-11708A) Telithromycin Cidal at 2X, 4X,8X (slow killing) S. mitis CEM-101 Cidal at 2X, 4X, 8X (112-1885A)Telithromycin Cidal at 2X, 4X, 8X Clarithromycin Cidal at 8X only (slowkilling) Azithromycin Cidal at 4X and 8X (slow killing)CEM-101 exhibited bactericidal activity when tested againstmacrolide-susceptible streptococci, CoNS and macrolide-resistantCLN-susceptible S. pneumoniae. CEM-101 MBC/MIC ratios can be high for S.aureus, but some strains showed MBC results remaining within thesusceptible range of concentrations.

Example

Activity on Chlamydia. CEM-101, TEL, AZI, CLR, and doxycycline wereprovided as powders and solubilized according to the instructions of themanufacturers. Drug suspensions were made fresh each time the assay wasrun.

C. pneumoniae: Isolates of C. pneumoniae tested included a referencestrain (TW 183), 9 isolates from children and adults with pneumonia fromthe United States (AR39, T2023, T2043, W6805, CWL 029, CM-1), an isolatefrom a child with pneumonia from Japan (J-21), and 2 strains frombronchoalveolar lavage specimens from patients with humanimmunodeficiency virus infection and pneumonia from the United States(BAL15 and BAL16).

C. trachomatis: 10 isolates of C. trachomatis, including standardisolates from the ATCC (E-BOUR, F-IC-CAL3, C-HAR32, J-UW-36, L2434,D-UW-57kx, B- HAR-36) and recent clinical isolates (N18(cervical),N19(cervical), 7015(infant eye))

In vitro susceptibility testing: Susceptibility testing of C. pneumoniaeand C. trachomatis was performed in cell culture using HEp-2 cells grownin 96-well microtiter plates. Each well was inoculated with 0.1 ml ofthe test strain diluted to yield 10³ to 10⁴ IFU/ per ml, centrifuged at1,700×g for 1 hr. and incubated at 35° C. for 1 hr. Wells were aspiratedand overlaid with 0.2 mL of medium containing 1 μg of cycloheximide permL and serial two-fold dilutions of the test drug.

Duplicate plates were inoculated. After incubation at 35° C. for 48-72hrs, cultures were fixed and stained for inclusions withfluorescein-conjugated antibody to the lipopolysaccharide genus antigen(Pathfinder, Kallestad Diagnostics, Chaska, Minn). The minimalinhibitory concentration (MIC) is the lowest antibiotic concentration atwhich no inclusions were seen. The minimal bactericidal concentration(MBC) was determined by aspirating the antibiotic containing medium,washing wells twice with phosphate buffered saline and addingantibiotic-free medium. Cultures were frozen at −70° C., thawed, passedonto new cells, incubated for 72 hrs then fixed and stained as above.The MBC is the lowest antibiotic concentration that results in noinclusions after passage. All tests were run in triplicate.

Activities of CEM-101 and Other Antibiotics Against 10 Isolates of C.Pneumoniae

MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 101 0.25-1.00.25 0.25 0.25-1.0 0.25 Telithromycin 0.015-0.25 0.06 0.06 0.015-0.250.06 Azithromycin  0.015-0.125 0.125 0.125  0.015-0.125 0.125Clarithromycin  0.015-0.125 0.06 0.06  0.015-0.125 0.06 Doxycycline0.015-0.06 0.06 0.06 0.015-0.06 0.06

Activities of CEM-101 and Other Antibiotics Against 10 Isolates of C.Trachomatis

MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 101 0.125-0.5 0.25 0.25 0.125-0.5  0.25 Telithromycin 0.015-0.25 0.06 0.06 0.015-0.250.06 Azithromycin  0.015-0.125 0.125 0.125  0.015-0.125 0.125Clarithromycin  0.015-0.125 0.06 0.06  0.015-0.125 0.06 Doxycycline0.015-0.06 0.06 0.06 0.015-0.06 0.06The results of this study demonstrated that CEM-101 has in vitroactivity against C. trachomatis and C. pneumoniae comparable to othermacrolides and ketolides.

Example

Tissue distribution. CEM-101 was well absorbed and distributed to thetissue. In the rat at 250 mg/kg/d, mean lung and liver concentrations ofCEM-101 were 17 and 15-fold higher than in plasma. Lung and liverconcentrations were 503 and 711-fold higher than plasma concentrationsat the 200 mg/kg/d dose in monkeys. Concentrations of CEM-101 in theheart were significantly lower than levels found in lung or liver withlevels 5 and 54-fold higher than plasma concentrations in rat andmonkey, respectively.

What is claimed is:
 1. A method for acute-exposure treatment of apatient exposed to one or more organisms selected from the groupconsisting of Bacillus anthracis and Burkholderia mallei, andcombinations thereof; the method comprising the step of administering tothe patient an effective amount of one or more compounds of the formula

or pharmaceutically acceptable salt thereof, wherein: R¹⁰ is hydrogen oracyl; X and Y are taken together with the attached carbon to formcarbonyl; V is C(O), C(═NR¹¹), CH(NR¹², R¹³), or N(R¹⁴)CH₂; where N(R¹⁴)is attached to the C-10 carbon; where R¹¹ is hydroxy or alkoxy; R¹² andR¹³ are each independently selected from the group consisting ofhydrogen, hydroxy, alkyl, alkoxy, heteroalkyl, aryl, arylalkyl,heteroaryl, and heteroarylalkyl, each of which is optionallysubstituted, and dimethylaminoalkyl, acyl, sulfonyl, ureido, andcarbamoyl; R¹⁴ is hydrogen, hydroxy, alkyl, alkoxy, heteroalkyl, aryl,arylalkyl, heteroaryl, or heteroarylalkyl, each of which is optionallysubstituted, or dimethylaminoalkyl, acyl, sulfonyl, ureido, orcarbamoyl; W is H, F, Cl, Br, I, or OH; A is CH₂, C(O), C(O)O, C(O)NH,S(O)₂, S(O)₂NH, or C(O)NHS(O)₂; B is C₀-C₁₀ alkylene, C₂-C₁₀ alkenylene,or C₂-C₁₀ alkynylene; and C is hydrogen, hydroxy, alkyl, alkoxy,heteroalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, each ofwhich is optionally substituted, or acyl, acyloxy, sulfonyl, ureido, orcarbamoyl; or a pharmaceutical composition thereof further comprisingone or more pharmaceutically acceptable carriers, diluents, orexcipients, or a combination thereof.
 2. A method for treating a patientwith a disease caused at least in part by one or more organisms selectedfrom the group consisting of Bacillus anthracis and Burkholderia mallei,and combinations thereof; the method comprising the step ofadministering to the patient an effective amount of one or morecompounds of the formula

or pharmaceutically acceptable salt thereof, wherein: R¹⁰ is hydrogen oracyl; X and Y are taken together with the attached carbon to formcarbonyl; V is C(O), C(═NR¹¹), CH(NR¹², R¹³), or N(R¹⁴)CH₂; where N(R¹⁴)is attached to the C-10 carbon; where R¹¹ is hydroxy or alkoxy; R¹² andR¹³ are each independently selected from the group consisting ofhydrogen, hydroxy, alkyl, alkoxy, heteroalkyl, aryl, arylalkyl,heteroaryl, and heteroarylalkyl, each of which is optionallysubstituted, and dimethylaminoalkyl, acyl, sulfonyl, ureido, andcarbamoyl; R¹⁴ is hydrogen, hydroxy, alkyl, alkoxy, heteroalkyl, aryl,arylalkyl, heteroaryl, or heteroarylalkyl, each of which is optionallysubstituted, or dimethylaminoalkyl, acyl, sulfonyl, ureido, orcarbamoyl; W is H, F, Cl, Br, I, or OH; A is CH₂, C(O), C(O)O, C(O)NH,S(O)₂, S(O)₂NH, or C(O)NHS(O)₂; B is C₀-C₁₀ alkylene, C₂-C₁₀ alkenylene,or C₂-C₁₀ alkynylene; and C is hydrogen, hydroxy, alkyl, alkoxy,heteroalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, each ofwhich is optionally substituted, or acyl, acyloxy, sulfonyl, ureido, orcarbamoyl; or a pharmaceutical composition thereof further comprisingone or more pharmaceutically acceptable carriers, diluents, orexcipients, or a combination thereof.
 3. The method of claim 1 whereinR¹⁰ is hydrogen.
 4. The method of claim 1 wherein A is CH_(2.)
 5. Themethod of claim 1 wherein B is (CH₂)_(n), where n is an integer from1-5.
 6. The method of claim 1 wherein C is aryl or heteroaryl, each ofwhich is optionally substituted.
 7. The method of claim 1 wherein C is3-aminophenyl or 3-pyridinyl.
 8. The method of claim 1 wherein V isC(O).
 9. The method of claim 1 wherein W is H or F.
 10. The method ofclaim 1 wherein W is F.
 11. The method of claim 1 wherein V is C(O); andW is F.
 12. The method of claim 1 wherein A is CH₂, B is (CH₂)_(n), andn is an integer from 2-4.
 13. The method of claim 12 wherein C is3-aminophenyl.
 14. The method of claim 13 wherein V is C(O).
 15. Themethod of claim 14 wherein W is F.
 16. The method of claim 15 wherein nis
 3. 17. The method of claim 16 wherein R¹⁰ is hydrogen.
 18. The methodof claim 1 wherein the compound is of the formula

or a pharmaceutically acceptable salt thereof.
 19. The method of claim 1wherein the compound is of the formula

where HX is a pharmaceutically acceptable salt forming acid.
 20. Themethod of claim 19 wherein HX is selected from the group consisting ofhydrochloric acid, tartaric acid, and combinations thereof.
 21. Themethod of claim 1 wherein the compound is of the formula


22. The method of claim 2 wherein the compound is of the formula

or a pharmaceutically acceptable salt thereof.
 23. The method of claim 2wherein the compound is of the formula

where HX is a pharmaceutically acceptable salt forming acid.
 24. Themethod of claim 23 wherein HX is selected from the group consisting ofhydrochloric acid, tartaric acid, and combinations thereof.
 25. Themethod of claim 2 wherein the compound is of the formula


26. The method of claim 18 wherein the organism is Bacillus anthracis.27. The method of claim 22 wherein the organism is Bacillus anthracis.